Biomedical subjects

Kang Zhang

Publications and source records attributed to Kang Zhang.

At least 37 records · Page 2Linked to original sources

PubMed2005

Loss of ER retention and sequestration of the wild-type ELOVL4 by Stargardt disease dominant negative mutants.

PURPOSE: Mutations in ELOVL4, a member of the fatty acid elongase (ELO) family, are responsible for autosomal dominant Stargardt-like macular degeneration. The specific role of ELOVL4 in photoreceptors and the degenerative events induced by dominant ELOVL4 mutations are not well understood. As a first step to identifying possible mechanisms contributing to cellular dysfunction, we transfected HEK293 and COS cells with fluorescent-labeled wild-type and mutant ELOVL4 constructs. Effects of mutant ELOVL4 on interaction with wild-type protein were examined in this in vitro model. METHODS: Wild-type and mutant ELOVL4 proteins including ELOVL4 truncation (270X, a truncated ELOVL4 protein at amino acid position 270) and ELOVL4 5 bp deletion (5bp-del) and ELOVL4 (5A, substituting the ER retention signal, KAKGD, with a five alanine amino acid tract) were expressed as EGFP or DsRed fusion proteins. Cellular localization of these proteins was examined by fluorescence microscopy. ELOVL4 protein aggregates were measured by co-immunoprecipitation and by sucrose gradient centrifugation followed by immunodetection with western blots. To study cellular status of cells expressing mutant ELOVL4 proteins, transfected cells were examined for upregulation of Bip and CHOP, markers for the unfolded protein response (UPR) by western blotting. RESULTS: ELOVL4 mutants were not retained within the ER but were rather mislocalized and formed aggregates. Importantly, when cotransfected with wild-type ELOVL4, the mutants bound to and sequestered the wild-type protein into the aggregates. Expression of ELOVL4 mutants also induced UPR as evidenced by Bip and CHOP expression. CONCLUSIONS: Using this in vitro cell system, we have identified alterations in wild-type ELOVL4 protein localization, aggregate formation, and the induction of cellular stress by the ELOVL4 mutants. We propose that "inactivation" of the wild-type ELOVL4 protein through sequestration to a non-ER compartment by ELOVL4 mutants may play a role in cellular dysfunction.

Animals↗

PubMed2005

Examination of ELN as a candidate gene in the Utah intracranial aneurysm pedigrees.

BACKGROUND AND PURPOSE: A study of intracranial aneurysm (IA) sibpairs suggested association of an ELN haplotype with IA risk. Subsequent linkage analysis of the ELN region on chromosome 7q11 in high-risk Utah IA pedigrees significantly confirmed linkage between IA and the ELN region. METHODS: We have investigated the ELN gene as a potential candidate gene for IA in Utah pedigrees. One IA case from each pedigree, who shared an ELN region haplotype segregating in the pedigree, was screened for mutation. The promoter region, 34 exons, and the 3'UTR (UnTranslated Region) of the ELN gene were screened for variants using DHPLC. RESULTS: Variants were observed in the promoter region, exons 4 and 6, and the 3'UTR. Variants in exon 6 and in one 3'UTR position were unique to Utah. The remaining variants were absent in the controls. There was no evidence for segregation of the ELN variants found in IA cases with the hypothesized chromosome 7 haplotypes segregating in pedigrees. CONCLUSIONS: Our analysis does not support ELN as the gene responsible for familial IA in the linked Utah IA pedigrees.

3' Untranslated Regions↗

PubMed2005

Autosomal dominant cone dystrophy caused by a novel mutation in the GCAP1 gene (GUCA1A).

PURPOSE: To describe the clinical features and genetic analysis of a family with an autosomal dominant cone dystrophy (adCD). METHODS: Selected members of a family with an autosomal dominant cone dystrophy underwent ophthalmic evaluation. Blood samples were obtained, genomic DNA was isolated, and genomic fragments were amplified by PCR. Linkage to locus D6S1017 was established. DHPLC mutational analysis and direct sequencing were used to identify a mutation in GUCA1A, the gene encoding the guanylate cyclase activating protein 1 (GCAP1). RESULTS: Of 24 individuals who are at risk of the disease in a five generation family, 11 members were affected. Clinical presentations included photophobia, color vision defects, central acuity loss, and legal blindness with advanced age. The disease phenotype was observed in the second and third decades of life and segregated in an autosomal dominant fashion. An electroretinogram performed on one proband revealed profoundly subnormal and prolonged photopic and flicker responses, but preserved scotopic ERGs, consistent with a cone dystrophy. Mutational analysis and direct sequencing revealed a C451T transition in GUCA1A, corresponding to a novel L151F mutation in GCAP1. Like the E155G mutation, this mutation occurs in the EF4 hand domain, a region of GCAP1 critical in conferring calcium sensitivity to the protein. The leucine at this position is highly conserved among vertebrate guanylate cyclase activating proteins. CONCLUSIONS: A novel L151F missense mutation in the EF4 high affinity Ca2+ binding site of GCAP1 is linked to adCD in a large pedigree. The cone dystrophy in this family shares clinical and electrophysiologic characteristics with other previously described adCD caused by mutations in GUCA1A.

Adult↗

PubMed2005

Mutation screen of the membrane-type frizzled-related protein (MFRP) gene in patients with inherited retinal degenerations.

MFRP is a member of the frizzled-related protein family and contains a cysteine-rich domain essential for Wnt binding and signaling. MFRP is highly expressed in the retinal pigment epithelial cells of the eye. A splice donor mutation in the mouse ortholog of Mfrp is responsible for photoreceptor degeneration in the rd6 mouse. For these reasons, we investigated MFRP as a candidate gene for a phenotype associated with mutations. We screened 152 patients with inherited retinal degenerations including retinitis pigmentosa, Leber congenital amaurosis and Stargardt macular dystrophy. We identified five polymorphisms in the 5' untranslated region, four missense changes, six isocoding variants and four intronic changes. None of the sequence variants were interpreted as pathogenic.

5' Untranslated Regions↗

PubMed2004

Mutant carbonic anhydrase 4 impairs pH regulation and causes retinal photoreceptor degeneration.

Retina and retinal pigment epithelium (RPE) belong to the metabolically most active tissues in the human body. Efficient removal of acid load from retina and RPE is a critical function mediated by the choriocapillaris. However, the mechanism by which pH homeostasis is maintained is largely unknown. Here, we show that a functional complex of carbonic anhydrase 4 (CA4) and Na+/bicarbonate co-transporter 1 (NBC1) is specifically expressed in the choriocapillaris and that missense mutations in CA4 linked to autosomal dominant rod-cone dystrophy disrupt NBC1-mediated HCO3- transport. Our results identify a novel pathogenic pathway in which a defect in a functional complex involved in maintaining pH balances, but not expressed in retina or RPE, leads to photoreceptor degeneration. The importance of a functional CA4 for survival of photoreceptors implies that CA inhibitors, which are widely used as medications, particularly in the treatment of glaucoma, may have long-term adverse effects on vision.

Carbonic Anhydrase IV↗

PubMed2004

Expression of wild type and mutant ELOVL4 in cell culture: subcellular localization and cell viability.

PURPOSE: ELOVL4 is a member of the fatty acid elongase (ELO) family of genes. Mutations of this gene are responsible for autosomal dominant Stargardt-like macular degeneration. However, the specific role of ELOVL4 in photoreceptor cells and the mechanism by which mutations in ELOVL4 causes macular degeneration are not known. In this study we examined the subcellular localization of wild type (wt) and mutant (mt) ELOVL4 EGFP fusion protein and the potential functional consequence of mtELOVL4 expression on cell viability. METHODS: Wt and mt ELOVL4 were expressed as EGFP fusion proteins in NIH 3T3 and HEK293 cells. Subcellular localizations of the fusion proteins were determined with a series of organelle-specific markers for endoplasmic reticulum (pDsRed2-ER), mitochondria (pDsRed2-Mito), peroxisomes (pDsRed2-Peroxi), and Golgi (BODIPY TR). Transfected cells were viewed using confocal and episcopic-fluorescence microscopy. Western blot analysis was performed to assess protein expression using an anti-GFP antibody. TUNEL staining was used to quantify apoptotic cell death. RESULTS: In cell transfection studies, wtELOVL4/EGFP fusion protein localized preferentially to the endoplasmic reticulum (ER) and was not found to be discernibly present in mitochondria, peroxisomes, or Golgi. In contrast, the truncated mutant fusion protein (which has no ER retention signal) appeared to be mislocalized to other compartments within transfected cells. Transfected cells expressing mtELOVL4/EGFP fusion protein exhibited induction of apoptotic cell death. CONCLUSIONS: Unlike wtELOVL4/EGFP fusion protein, the mtELOVL4/EGFP fusion protein did not localize to the ER but rather appeared to be sequestered elsewhere in an aggregated pattern in the cytoplasm. The apoptosis induced by the mutant ELOVL4 fusion protein may be the mechanism whereby photoreceptor cells degenerate in Stargardt-like macular degeneration. Our study has provided an important in vitro model system for further assessment of ELOVL4 biochemical functions.

Animals↗

PubMed2004

Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.

Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.

Animals↗

PubMed2004

Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Autosomal dominant FEVR is genetically heterogeneous, but its principal locus, EVR1, is on chromosome 11q13-q23. The gene encoding the Wnt receptor frizzled-4 (FZD4) was recently reported to be the EVR1 gene, but our mutation screen revealed fewer patients harboring mutations than expected. Here, we describe mutations in a second gene at the EVR1 locus, low-density-lipoprotein receptor-related protein 5 (LRP5), a Wnt coreceptor. This finding further underlines the significance of Wnt signaling in the vascularization of the eye and highlights the potential dangers of using multiple families to refine genetic intervals in gene-identification studies.

Amino Acid Sequence↗

PubMed2004

Dominant cone and cone-rod dystrophies: functional analysis of mutations in retGC1 and GCAP1.

The regulation of cGMP levels is central to the normal process of phototransduction in both cone and rod photoreceptor cells. Two of the proteins involved in this process are the enzyme, retinal guanylate cyclase (retGC), and its activating protein (GCAP) through which activity is regulated via changes in cellular Ca2+ levels. Dominant cone-rod dystrophies arising from changes in retGC1 are essentially restricted to mutations in codon 838 and result in the replacement of a conserved arginine residue with either cysteine, histidine or serine. In all three cases, the effect of the substitution on the in vitro cyclase activity is a loss of Ca2+ sensitivity arising from an increased stability of the coiled-coil domain of the protein dimer and retention of cyclase activity. In contrast, mutations in the Ca2+-coordinating EF hands of GCAP1 result in dominant cone dystrophy; the consequences of these mutations is a reduced ability of the mutant protein to regulate retGC activity in response to changes in Ca2+ levels. Functionally therefore, the retGC2 and GCAP2 mutations are similar in reducing the feedback inhibition of Ca2+ on cyclase activity and thereby on cGMP levels in the photoreceptors.

Calcium↗

PubMed2004

Dominant optic atrophy, sensorineural hearing loss, ptosis, and ophthalmoplegia: a syndrome caused by a missense mutation in OPA1.

PURPOSE: To describe the clinical features of and identify the disease-causing mutation in a large Utah family segregating a dominantly inherited syndrome of optic atrophy, sensorineural hearing loss, ptosis, and ophthalmoplegia. DESIGN: Observational case series. METHODS: Thirty individuals at risk for a syndrome of optic atrophy, sensorineural hearing loss, ptosis, and ophthalmoplegia in a single family underwent clinical examinations and venipuncture. Linkage analysis and mutation screening of the optic atrophy 1 gene (OPA1) were performed. RESULTS: Eighteen individuals demonstrated characteristics of the syndrome. Genetic analysis identified a G-->A substitution at nucleotide position 1334 in exon 14 of OPA1 causing an arginine-to-histidine change (R445H) in all affected members of the family. This change segregated with the disease phenotype in the study family with a LOD score of 7.02 at theta; = 0 and was not found in 200 normal control subjects. Analysis of an unrelated Belgian family with a similar phenotype revealed the same R445H mutation segregating with the disease phenotype. CONCLUSIONS: This study describes a mutation in OPA1 causing a unique syndrome of optic atrophy, sensorineural hearing loss, ptosis, and ophthalmoplegia. These results expand the spectrum of human disease associated with mutations of OPA1 and indicate that ophthalmologists caring for patients with optic atrophy should inquire about possible associated hearing loss. Although OPA1 is a nuclear gene, the gene product localizes to mitochondria, suggesting that mitochondrial dysfunction may be the final common pathway for many forms of syndromic and nonsyndromic optic atrophy, hearing loss, and external ophthalmoplegia.

Adolescent↗

PubMed2004

A novel RDS/peripherin gene mutation associated with diverse macular phenotypes.

Pattern dystrophy is a heterogeneous group of retinal dystrophies of which butterfly-shaped pattern dystrophy (BPD) and adult-onset foveomacular dystrophy (AOFMD) are the two most common forms. BPD is characterized by a butterfly-shaped, irregular, depigmented lesion at the level of the retinal pigment epithelium. In contrast, AOFMD is characterized by the presence of slightly elevated, symmetric, solitary, round to oval, yellow lesions at the level of the retinal pigment epithelium. We identified three independent kindreds with pattern dystrophy, one with four patients affected with BPD and the other two with 14 affected patients with AOFMD. We performed complete ophthalmic examination, fluorescein angiography, linkage mapping, and mutational screening in the RDS/peripherin gene in the affected patients. Patients affected with BPD had a best-corrected vision of 20/20 to 20/25, whereas vision in the eyes of patients with AOFMD ranged from 20/20 to 20/400. In all three kindreds, sequence analysis identified an A-to-G change at nucleotide position 422 of the RDS/peripherin gene, predicting a novel Tyr-141-Cys substitution. A haplotype analysis revealed that these three kindreds shared an identical disease haplotype at the RDS/peripherin locus, indicating that the mutation reflects a founder effect. The sequence change that segregated with the disease phenotype was not observed in 200 control chromosomes. Our results identified a novel mutation in the RDS/ peripherin gene that can cause diverse macular phenotypes. Genetic and clinical investigation of pattern dystrophy may provide useful diagnostic tools and new treatment strategies for this disorder.

Adult↗

PubMed2004

Spectrum and frequency of FZD4 mutations in familial exudative vitreoretinopathy.

PURPOSE: Mutations in the frizzled-4 gene (FZD4) have recently been associated with autosomal dominant familial exudative vitreoretinopathy (FEVR) in families linking to the EVR1 locus on the long arm of chromosome 11. The purpose of this study was to screen FZD4 in a panel of 40 patients with FEVR to identify the types and location of mutations and to calculate what proportion of this heterogeneous condition is attributable to FZD4 mutations. METHODS: PCR products were generated from genomic DNA with primers designed to amplify the coding sequence of FZD4. The PCR products were screened for mutations by single-strand conformational polymorphism-heteroduplex analysis (SSCP-HA) and by direct sequencing. RESULTS: In total, eight mutations were identified, seven of which were novel. Three were deletions (c957delG, c1498delA, and c1501-1502delCT), one was a nonsense mutation (Q505X), and four were missense mutations (G36D, M105T, M157V, and S497F). CONCLUSIONS: Eight mutations have been identified in the FZD4 gene in a cohort of 40 unrelated patients with FEVR. This result indicates that FZD4 mutations are responsible for only 20% of FEVR index cases and suggests that the other FEVR loci may account for more cases than previously anticipated.

Adult↗

PubMed2004

A novel mutation in the ELOVL4 gene causes autosomal dominant Stargardt-like macular dystrophy.

PURPOSE: To conduct clinical and genetic studies in a European family with autosomal dominant Stargardt-like macular dystrophy (adSTGD-like MD) and to investigate the functional consequences of a novel ELOVL4 mutation. METHODS: Ophthalmic examination and mutation screening by direct sequencing of the ELOVL4 gene was performed in two affected individuals. Wild-type and mutant ELOVL4 genes were expressed as enhanced green fluorescent protein (EGFP) fusion proteins in transient transfection in NIH-3T3 and HEK293 cells. To determine the subcellular localization of ELOVL4, an endoplasmic-reticulum (ER)-specific marker for pDsRed2-ER was cotransfected with ELOVL4 constructs. Transfected cells were viewed by confocal microscopy. Western blot analysis was performed to assess protein expression using an anti-GFP antibody. RESULTS: Affected patients exhibited macular atrophy with surrounding flecks characteristic of adSTGD-like MD. A novel ELOVL4 p.Tyr270X mutation was detected in affected individuals. In cell-transfection studies, wild-type ELOVL4 localized preferentially to the ER. In contrast, the mutant protein appeared to be mislocalized within transfected cells. CONCLUSIONS: In a European family with adSTGD-like MD, a novel ELOVL4 mutation was found to underlie the disorder. Transfection studies indicated that, unlike wild-type ELOVL4, the mutant protein does not localize to the ER but rather appears to be sequestered elsewhere in an aggregated pattern in the cytoplasm. Further analysis of the function of normal and mutant ELOVL4 will provide insight into the mechanism of macular degeneration.

Adolescent↗

PubMed2003

Elovl4 mRNA distribution in the developing mouse retina and phylogenetic conservation of Elovl4 genes.

PURPOSE: Stargardt-like macular dystrophy (STGD3) is an autosomal dominant form of early onset macular degeneration. The disease causing gene ELOVL4 encodes a protein that belongs to a family of proteins functioning in elongation of long chain fatty acids. The purpose of this study is to characterize cross-species conservation of ELOVL4 and investigate its mRNA distribution in the developing mouse eye. METHODS: Bovine and porcine orthologs of the human ELOVL4 gene were cloned using RT-PCR method. EST and HTGS databases were searched for orthologs of ELOVL4. Cross-species alignments were performed using ClustalW. In situ hybridizations using murine Elovl4 probes were performed on frozen sections of mouse eyes. RESULTS: Elovl4 orthologs from mammalian to invertebrate species share strong sequence homology with human ELOVL4 at the amino acid level, suggesting functional conservation of Elovl4 during evolution. Expression of Elovl4 in mouse retina begins at E15 during embryogenesis and persists in postnatal stages. However, Elovl4 is predominantly expressed in the retinal ganglion cells at P1-P3, followed by predominant expression in the outer nuclear layer at P7, with its final expression enriched in inner segments of photoreceptors. CONCLUSIONS: Elovl4 expression in developing retina follows a dynamic pattern. It switches from predominant ganglion cell expression in embryonic and early postnatal development to predominant expression in the photoreceptor inner segments in later stages. Phylogenetic analysis reveals strong conservation of Elovl4 among different species throughout the vertebrate subphylum consistent with our hypothesis that ELOVL4 performs a fundamentally important function.

Amino Acid Sequence↗

PubMed2003

A novel mutation in the RDS/Peripherin gene causes adult-onset foveomacular dystrophy.

PURPOSE: To describe a novel mutation in the RDS/Peripherin gene that results in a moderately severe form of adult-onset foveomacular dystrophy. DESIGN: Observational case series. METHODS: Selected members of a family with adult-onset foveomacular dystrophy underwent complete ophthalmic evaluation, including fundus photography and fluorescein angiography, in a tertiary care referral center. The study population consisted of 12 members of a Caucasian kindred. After providing informed consent, patients donated blood for genomic DNA extraction and mutational screening using standard techniques. The main outcome measure were the presence of a RDS/Peripherin gene mutation in a patient with the disease and its absence in unaffected family members and controls. RESULTS: Eight affected family members and no unaffected family members demonstrated a single guanine base deletion at nucleotide 112 that led to premature termination at amino acid 38 of RDS/Peripherin polypeptide. This frameshift mutation results in truncation of nearly 90% of the gene product, thus probably representing a null allele. That results in a relatively severe phenotype, with choroidal neovascularization developing in two patients and geographic atrophy involving the macula in three patients. CONCLUSIONS: We describe a frameshift null mutation in the RDS/Peripherin gene associated with a relatively severe manifestation of adult-onset foveomacular dystrophy in affected family members.

Adult↗

PubMed2003

Genomic organisation and alternative splicing of human RIM1, a gene implicated in autosomal dominant cone-rod dystrophy (CORD7).

A mutation has been identified in the Rab3A-interacting molecule (RIM1) gene in CORD7, an autosomal dominant cone-rod dystrophy that localises to chromosome 6q14. The G to A point mutation results in an Arg844His substitution in the C(2)A domain of the protein that segregates with disease. This mutation is absent in over 200 control chromosomes, indicating that it is not a common polymorphism, and the almost complete sequence conservation of the C(2)A domain between human and rat RIM1 is consistent with a disease role for the change. RIM1 is expressed in brain and photoreceptors of the retina where it is localised to the pre-synaptic ribbons in ribbon synapses. The RIM1 gene is composed of at least 35 exons, spans 577 kb of genomic DNA, and encodes a protein of up to 1693 residues. The transcript shows extensive alternative splicing involving exons 17, 21-26 and 28-30.

Alternative Splicing↗

PubMed2003

Suppression of the ELO-2 FA elongation activity results in alterations of the fatty acid composition and multiple physiological defects, including abnormal ultradian rhythms, in Caenorhabditis elegans.

While the general steps of fatty acid (FA) biosynthesis are well understood, the individual enzymes involved in the elongation of long chain saturated and polyunsaturated FA (PUFA) are largely unknown. Recent research indicates that these enzymes might be of considerable physiological importance for human health. We use Caenorhabditis elegans to study FA elongation activities and associated abnormal phenotypes. In this article we report that the predicted C. elegans F11E6.5/ELO-2 is a functional enzyme with the FA elongation activity. It is responsible for the elongation of palmitic acid and is involved in PUFA biosynthesis. RNAi-mediated suppression of ELO-2 causes an accumulation of palmitate and an associated decrease in the PUFA fraction in triacylglycerides and phospholipid classes. This imbalance in the FA composition results in multiple phenotypic defects such as slow growth, small body size, reproductive defects, and changes in rhythmic behavior. ELO-2 cooperates with the previously reported ELO-1 in 20-carbon PUFA production, and at least one of the enzymes must function to provide normal growth and development in C. elegans. The presented data indicate that suppression of a single enzyme of the FA elongation machinery is enough to affect various organs and systems in worms. This effect resembles syndromic disorders in humans.

Acetyltransferases↗

PubMed2002

Mutations in the RPGR gene cause X-linked cone dystrophy.

X-linked cone dystrophy is a type of hereditary retinal degeneration characterized by a progressive dysfunction of the day vision or photopic (cone) system with preservation of night vision or scotopic (rod) function. The disease presents with a triad of photophobia, loss of color vision and reduced central vision. This phenotype is distinct from retinitis pigmentosa (RP) in which there are prominent night and peripheral vision disturbances. X-linked cone dystrophy is a genetically heterogeneous disorder, with linkage to loci on Xp11.4--Xp21.1 (COD1, OMIM 304020) and Xq27 (COD2, OMIM 303800). COD1 maps to a region that harbors the RPGR gene, mutations in which account for >70% of patients with X-linked RP. The majority of these mutations reside in one purine-rich exon, ORF15, encoding 567 amino acids with a repetitive domain rich in glutamic acid residues. We mapped two families with X-linked cone dystrophy to the COD1 locus and identified two distinct mutations in ORF15 in the RPGR gene (ORF15+1343_1344delGG and ORF15+694_708del15) leading to a frame-shift and premature termination of translation in one case and a deletion of five amino acids in another. Consistent with expression of RPGR in rods and cones, our results show that mutations in RPGR, in addition to X-linked RP, can also cause cone-specific degeneration.

Amino Acid Sequence↗