Biomedical subjects

Jochen Graw

Publications and source records attributed to Jochen Graw.

34 records · Page 2Linked to original sources

PubMed2004

Mutation analysis of congenital cataracts in Indian families: identification of SNPS and a new causative allele in CRYBB2 gene.

PURPOSE: To study some functional candidate genes in cataract families of Indian descent. METHODS: Nine Indian families, clinically documented to have congenital/childhood cataracts, were screened for mutations in candidate genes such as CRYG (A-->D), CRYBB2, and GJA8 by PCR analyses and sequencing. Genomic DNA samples of either probands or any representative affected member of each family were PCR amplified and sequenced commercially. Documentation of single nucleotide polymorphisms (SNPs) and candidate mutations was done through BLAST SEARCH (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?). RESULTS: Several single nucleotide polymorphisms in CRYG, CRYBB2, and GJA8 genes were observed. Because they do not co-segregate with the phenotype, they were excluded as candidates for the cataract formation in these patients. However, a substitution (W151C in exon 6 of CRYBB2) was identified as the most likely causative mutation underlying the phenotype of central nuclear cataract in all affected members of family C176. Protein structural interpretations demonstrated that no major structural alterations could be predicted and that even the hydrogen bonds to the neighboring Leu166 were unchanged. Surprisingly, hydropathy analysis of the mutant betaB2-crystallin featuring the amino acids at position 147 to 155, further increased the hydrophobicity, which might impair the solubility of the mutant protein. Finally, the Cys residue at position 151 might possibly be involved in intramolecular disulphide bridges with other cysteines during translation, possibly leading to dramatic structural changes. CONCLUSIONS: Exon 6 of CRYBB2 appears to be a critical region susceptible for mutations leading to lens opacity.

Adolescent↗

PubMed2004

Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3.

The mesencephalic dopamine (mesDA) system is involved in the control of movement and behavior. The expression of Pitx3 in the brain is restricted to the mesDA system and the gene is induced relatively late, at E11.5, a time when tyrosine hydroxylase (Th) gene expression is initiated. We show here that, in the Pitx3-deficient aphakia (ak) mouse mutant, the mesDA system is malformed. Owing to the developmental failure of mesDA neurons in the lateral field of the midbrain, mesDA neurons are not found in the SNc and the projections to the caudate putamen are selectively lost. However, Pitx3 is expressed in all mesDA neurons in control animals. Therefore, mesDA neurons react specifically to the loss of Pitx3. Defects of motor control where not seen in the ak mice, suggesting that other neuronal systems compensate for the absence of the nigrostriatal pathway. However, an overall lower activity was observed. The results suggest that Pitx3 is specifically required for the formation of the SNc subfield at the onset of dopaminergic neuron differentiation.

Animals↗

PubMed2004

Congenital hereditary cataracts.

Congenital cataracts are rare and occur in developed countries with a frequency of 30 cases among 100,000 births with a further 10 cases being diagnosed during childhood. They reflect mainly genetically caused developmental alterations in the lens and surrounding ocular tissues. Even if modern Human Genetics has made large steps forward in the characterization of human hereditary disorders, the underlying developmental processes can only be investigated in model organisms. The mouse is such a good model because of its similarity (as a mammal) and its genetic characterization. This review brings together our genetic and developmental knowledge of congenital, human cataracts with the corresponding mouse models. First, early events will be influenced by genes coding for transcription factors like Pax6, Pitx3, Maf or Sox. If the lens is maturing, mutations affecting the lens membranes (aquaporins/Mip, Lim-2 or connexins) or the structural proteins of the cytosol of the lens fiber cells (the crystallins) become more important. From a genetic point of view it becomes obvious that cataract-causing mutations are not distributed randomly. The discovery of a broad variety of genes important for eye and lens development made much progress in the recent years. Nevertheless, there still remains a long list of mutations to be characterized and functionally investigated both in mouse and man indicating a broad genetic heterogeneity in that which clinicians simply refer to as a "cataract".

Alleles↗

PubMed2003

The genetic and molecular basis of congenital eye defects.

The mature eye is a complex organ that develops through a highly organized process during embryogenesis. Alterations in its genetic programming can lead to severe disorders that become apparent at birth or shortly afterwards; for example, one-half of the cases of blindness in children have a genetic cause. This review outlines the genetic basis of eye development, as determined by mutation analysis in patients and in model organisms. A better understanding of how this intricate organ develops at the genetic and cellular level is central to our understanding of the pathologies that afflict it.

Animals↗

PubMed2003

Developmental genetics in ophthalmology.

Much of our knowledge about the function of genes in mammalian development has been derived from the molecular analysis of spontaneous or induced mutations in the mouse. Since mutations affecting the mouse eye can be easily identified, a remarkable number of mutant lines provide animal models for congenital anomalies in man. To understand the mechanisms of lens development in detail, the isolation of the corresponding genes and the characterization of the mutations at the molecular level are important. A prerequisite for molecular analysis is the chromosomal localization of the gene. In this review, some mutants from our institute will be discussed according to the embryological time scale of the expression of the affected genes, reflecting also their genetic hierarchy. (1) In the aphakia mouse mutant, two deletions in the promoter of the homeobox transcription factor Pitx3 lead to a loss of its function and to an arrest of eye development at the lens stalk stage. Mutations in the homologous human PITX3 gene have been demonstrated to be causative of cataracts and the dysmorphology of the anterior segment of the eye. (2) Connexin50 is present in the lens vesicle. Later on, it becomes abundant in the anterior part of the fiber cells and in the lens epithelial cells. Mutations in the connexin50-encoding gene Gja8 lead to dominant cataracts. (3) alphaA-crystallin is present in the mouse lens cup, in the posterior half of the lens vesicle, and later in a high concentration in the lens fiber cells. Mutations in the alphaA-crystallin-encoding gene Cryaa lead to recessive and dominant cataracts. (4) Mutations in the gamma-crystallin -encoding genes (Cryg) are the most frequent cause of congenital, dominant nuclear, or total cataracts in the mouse. Indications from our first studies in congenital human cataracts support these data. (5) Some postnatal, progressive cataracts have been characterized by mutations in the beta-crystallin -encoding genes (Cryb). Since at least one of them is also expressed in the retina and the brain, effects on these tissues have to be considered, too.

Animals↗

PubMed2003

Mutation in intron 6 of the hamster Mitf gene leads to skipping of the subsequent exon and creates a novel animal model for the human Waardenburg syndrome type II.

In the course of analysis of ENU-induced mutations in Syrian hamsters, a novel dominant anophthalmic white mutant (Wh(V203)) with hearing loss was recovered. Because of this phenotype and a close linkage to the Tpi gene, the Mitf gene was considered as a candidate gene. In the Mitf cDNA, a deletion of 76 bp covering the entire exon 7 was detected. Further molecular analysis revealed a T --> A exchange 16 bp upstream of the end of intron 6, leading to skipping of exon 7. These 16 bp at the end of intron 6 are identical in hamster, rat, mouse, and humans, indicating high conservation during evolution and a functional importance in splicing. Since the loss of exon 7 changes the open reading frame of the MITF transcript, translation will be stopped after 10 new amino acids. The truncated protein is predicted to contain only a part of the basic region and will miss the two helical domains and the leucine zipper. The Wh(V203) mutation in the Syrian hamster affects the same functional domains of the Mitf transcription factor as the human R124X mutation, causing human Waardenburg syndrome type II. Therefore, the Wh(V203) hamster mutant provides a novel model for this particular syndrome.

Amino Acid Sequence↗

PubMed2003

An in vivo doxycycline-controlled expression system for functional studies of the retina.

PURPOSE: Transgenic mice were developed that express tetracycline-controlled transactivator 1 (tTA1) specifically in photoreceptor cells. In these mice the transcription of the gene of interest can be easily inactivated in the retina in a short time frame. METHODS: A construct was prepared containing tTA1 under control of the murine rhodopsin regulatory region. This construct was used for the generation of transgenic mice. In situ hybridization was performed to study the distribution of the transactivator in the retina. The activity of the transactivator was analyzed by mating the lines with a luciferase reporter transgenic mouse. tTA1 activity and doxycycline's ability to block it were analyzed by luciferase assay. The effects of tTA1 on the retina were assessed by histology and electrophysiology. RESULTS: Two transgenic lines were developed that specifically express tTA1 in photoreceptor cells. The time course of transgene expression replicated transcription of endogenous rhodopsin. tTA1 was not toxic to the retina. Transactivator activity was blocked readily by doxycycline. CONCLUSIONS: An expression system for photoreceptor cells was generated to drive transcription in a cell-specific and time-controllable manner. This system is suitable for the study of factors involved in retinal biology and of mutant forms of genes involved in retinal diseases.

Animals↗

PubMed2002

Altered aggregation properties of mutant gamma-crystallins cause inherited cataract.

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gammaB-crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant gamma-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.

Amyloid↗

PubMed2002

Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype.

Pax6 is a key regulator of eye development in vertebrates and invertebrates, and heterozygous loss-of-function mutations of the mouse Pax6 gene result in the Small eye phenotype, in which a small lens is a constant feature. To provide an understanding of the mechanisms underlying this haploinsufficient phenotype, we evaluated in Pax6 heterozygous mice the effects of reduced Pax6 gene dosage on the activity of other transcription factors regulating eye formation. We found that Six3 expression was specifically reduced in lenses of Pax6 heterozygous mouse embryos. Interactions between orthologous genes from the Pax and Six families have been identified in Drosophila and vertebrate species, and we examined the control of Pax6 and Six3 gene expression in the developing mouse lens. Using in vitro and transgenic approaches, we found that either transcription factor binds regulatory sequences from the counterpart gene and that both genes mutually activate their expression. These studies define a functional relationship in the lens in which Six3 expression is dosage-dependent on Pax6 and where, conversely, Six3 activates Pax6. Accordingly, we show a rescue of the Pax6 haploinsufficient lens phenotype after lens-specific expression of Six3 in transgenic mice. This phenotypic rescue was accompanied by cell proliferation and activation of the platelet-derived growth factor alpha-R/cyclin D1 signaling pathway. Our findings thus provide a mechanism implicating gene regulatory interactions between Pax6 and Six3 in the tissue-specific defects found in Pax6 heterozygous mice.

Animals↗

PubMed2002

Recessive transmission of a multiple endocrine neoplasia syndrome in the rat.

We describe a novel hereditary cancer syndrome in the rat that is transmitted by a recessive gene mutation. Animals exhibiting the mutant phenotype develop multiple neuroendocrine malignancies within the first year of life. The endocrine neoplasia is characterized by bilateral adrenal pheochromocytoma, multiple extra-adrenal pheochromocytoma, bilateral medullary thyroid cell neoplasia, bilateral parathyroid hyperplasia, and pituitary adenoma. The appearance of neoplastic disease is preceded by the development of bilateral juvenile cataracts. Although the spectrum of affected tissues is reminiscent of human forms of multiple endocrine neoplasia (MEN), no germ-line mutations were detected in the Ret or Menin genes that are responsible for the dominantly inherited MEN syndromes in humans. Segregation studies in F1 and F2 crosses yielded frequencies of affected animals entirely consistent with a recessive autosomal mode of inheritance. The lack of the phenotype in F1 animals effectively excludes a germ-line tumor suppressor gene mutation as the causal event. The absence of mutation of known MEN genes and the unique constellation of affected tissues, plus the recessive mode of inheritance, lead us to conclude that the mutation of an as yet unknown gene is responsible for this syndrome of inherited neuroendocrine cancer.

Animals↗

PubMed2002

V76D mutation in a conserved gD-crystallin region leads to dominant cataracts in mice.

During a large-scale ENU mutagenesis screen, a mouse mutant with a dominant cataract was detected and referred to as Aey4. Aim of this study was the morphological description of the mutant, the mapping of the mutation, and the characterization of the underlying molecular lesion. The slit-lamp examination revealed a strong nuclear cataract surrounded by a homogeneous milky opacity in the inner cortex. The histological analysis demonstrated remnants of cell nuclei throughout the entire lens. The mutation was mapped to Chromosome 1 by a genome-wide linkage making the six gamma-crystallin encoding genes and the closely linked betaA2-crystallin encoding gene to relevant candidate genes. Finally, a T-->A exchange in exon 2 of the gammaD-crystallin encoding gene (symbol: Crygd) was demonstrated to be causative for the cataract phenotype; this particular mutation is, therefore, referred to Crygo(Aey4). The alteration in codon 76 leads to an amino acid exchange of Val-->Asp. Val at this position is highly conserved; it is found in all mouse and rat gammaD/E/F-crystallins as well as in the human gammaA- and gammaD-crystallins. It may be replaced solely by Ile, which is present in all bovine gamma-crystallins, in the rat and mouse gammaA/B/C-crystallins, as well as in the human gammaB/C-crystallins. It is predicted that the exchange of a hydrophobic side chain by a polar and acidic one might influence the microenvironment by a dramatic decrease of the isoelectric point by 1.5 pH units in the 10 amino acids surrounding position 76. The Crygd(Aey4) additionally demonstrates the importance of the integrity of the Cryg gene cluster for lens transparency.

Amino Acid Substitution↗

PubMed2002

Ethylnitrosourea-induced base pair substitution affects splicing of the mouse gammaE-crystallin encoding gene leading to the expression of a hybrid protein and to a cataract.

A novel ENU-induced mutation in the mouse leading to a nuclear and cortical opacity of the eye lens (ENU418) was mapped to proximal chromosome 1 by a genome-wide mapping approach. It suggests that the cluster of gamma-crystallin encoding genes (Cryg) and the betaA2-crystallin encoding gene Cryba2 are excellent candidate genes. An A --> G exchange in the middle of intron 1 of the Cryge gene was found as the only alteration cosegregating with the cataractous phenotype. The mutation was confirmed by the presence of a novel restriction site for ApaI in the corresponding genomic DNA fragment. The mutation represses splicing of intron 1; the additional 92 bp in the corresponding cDNA leads to a frameshift and the expression of a novel hybrid protein containing 3 amino acids of the gammaE-crystallin at the N terminus, but 153 novel amino acids. The Cryge(ENU418) protein has a calculated molecular mass of approximately 15.6 kD and an alkaline isoelectric point (pH 10.1) and is predicted to have two hydrophobic domains. Western blot analysis using a polyclonal antibody against the hydrophilic C-terminal part of the Cryge(ENU418)-specific protein demonstrated its stable expression in the cataractous lenses; it was not found in the wild types. Histological analysis of the cataractous lenses indicated that the expression of the new protein disrupts the cellular structure of the eye lens.

Alkylating Agents↗

PubMed2002

A 6-bp deletion in the Crygc gene leading to a nuclear and radial cataract in the mouse.

PURPOSE: A mouse mutant expressing a bilateral nuclear and radial cataract was found after paternal treatment with chlorambucil. The purpose of this study was to establish the linkage of the mutation to a particular chromosome to allow molecular characterization. Moreover, the mutants were examined morphologically. METHODS: Isolated lenses were photographed and histologic sections of the eye were analyzed according to standard procedures. The mutation was localized to chromosome 1 by allelism testing with the Cryge(nz) mutation. Candidate genes were amplified by PCR from cDNA or genomic DNA and sequenced. RESULTS: A novel mouse cataract was characterized by a nuclear and radial opacification of the lens. The lenses of the mutants are smaller than those of the wild type. The histologic analysis demonstrated degeneration of lens fibers in the lens core. Abnormal remnants of cell nuclei are present throughout the entire lens. Genetic analysis revealed allelism to the Cat2 group of dominant cataracts on mouse chromosome 1; therefore, the cluster of the Cryg genes and the closely linked Cryba2 gene were tested as candidates. A 6-bp deletion in exon 3 of the gammaC-crystallin encoding gene (Crygc) is causative for the cataract phenotype; the mutation is therefore designated CrygcChl3. The deletion of the bases 420 to 425 leads to a loss of two amino acids, Gly and Arg, in the fourth Greek-key motif. CONCLUSIONS: The CrygcChl3 is the first mutation in the mouse affecting the Crygc gene. Dominant mutations for five of the six Cryg genes on mouse chromosome 1 have now been characterized, demonstrating the importance of this gene cluster for lens transparency.

Alleles↗

PubMed2002

Altered Na,K-ATPase pattern in gamma-crystallin mutant mice.

PURPOSE: Na,K-adenosine triphosphatase (ATPase) activity is elevated in the lenses of murine cataract Cryge(t) and Cryge(ns) mutant mice. In the present study, the expression of Na,K-ATPase alpha1, alpha2, and alpha3 catalytic subunit polypeptides was examined in the lenses of these mutant mice. METHODS: Membrane material was isolated from lenses and brain of 3-week-old wild-type mice, as well as heterozygous and homozygous mutant mice. Microsomal membranes were prepared by centrifugation of the homogenized material, and Na,K-ATPase polypeptides were detected by immunoblot analysis with antibodies directed against the Na,K-ATPase isoforms alpha1, alpha2, and alpha3. RESULTS: For the Na,K-ATPase isoforms alpha2 and alpha3, membrane material obtained from the homozygous cataract mutants showed dense immunoblot bands that were not detected in material obtained from wild-type mice. An apparent increase of the alpha1 Na,K-ATPase isoform band density was also detected in lens material from the homozygous mutant mice. The Na,K-ATPase alpha3 polypeptide was also detected in lens membrane material obtained from heterozygous mice of both mutant strains. The alpha2 Na,K-ATPase polypeptide was observed in lens membrane material obtained from heterozygous Cryge(t) mice, and a less dense band was detected in heterozygous Cryge(ns) mice. Band densities of Na,K-ATPase subunits alpha1, alpha2, and alpha3 detected in brain membrane material were similar in both mutant and wild-type mice. CONCLUSIONS: The immunoblot results suggest that the abundance of Na,K-ATPase polypeptide is increased in the lens of the cataract mouse mutant but is not altered in the brain. The expression of the alpha2 and alpha3 isoform proteins of Na,K-ATPase is markedly upregulated in the cataractous lens.

Animals↗

PubMed2002

Crygf(Rop): the first mutation in the Crygf gene causing a unique radial lens opacity.

PURPOSE: The Rop (radial opacity) mutation, which was recovered in a mutagenicity screen after paternal treatment with procarbazine, was analyzed to determine phenotype, chromosomal localization, candidate genes, and molecular lesion. METHODS: Native lenses were photographed under a dissecting microscope. Histologic sections of the eye were made according to standard procedures. Fine mapping of the mutation in relation to microsatellite markers for mouse chromosome 1 was performed. Candidate genes were amplified by PCR from cDNA or genomic DNA and sequenced. RESULTS: The nuclear opacity of the heterozygous mutants showed radial structures, whereas the opacity of the homozygotes was homogenous. The histologic analysis revealed changes in the lens nucleus, which corresponds to the pronounced opacification in lenses of homozygous mutants. The allelism of Rop to the Cat2 group of dominant cataracts on mouse chromosome 1 was confirmed by linkage to microsatellite markers D1Mit156 and D1Mit181. The cluster of the Cryg genes and the closely linked Cryba2 gene were tested as candidates. A T-->A exchange in exon 2 of the Crygf gene leads to a Val-->Glu exchange in codon 38 and was considered to be causative for the cataract phenotype; therefore, Crygf(Rop) has been suggested as the designation for the mutation. CONCLUSIONS: Crygf(Rop) is the first mutation affecting the Crygf gene. Dominant cataract mutations for all six Cryg genes on mouse chromosome 1 have now been characterized, demonstrating the importance of this gene cluster in lens transparency.

Animals↗

PubMedDate not supplied

Further genetic heterogeneity for autosomal dominant human sutural cataracts.

A unique sutural cataract was observed in a 4-generation German family to be transmitted as an isolated autosomal, dominant trait. Since mutations in the gamma-crystallin encoding CRYG genes have previously been demonstrated to be the most frequent reason for isolated congenital cataracts, all 4 active CRYG genes have been sequenced. A single base-pair change in the CRYGA gene has been shown, leading to a premature stop codon. This was not observed in 170 control individuals. However, it did not segregate with the disease phenotype. This is the first truncating mutation in an active CRYG gene without a dominant phenotype. As the CRYGA mutation did not explain the cataract, several other candidate loci (CCV, GJA8, CRYBB2, BFSP2, MIP, GJA8, CENTRAL POUCH-LIKE, CRYBA1) were investigated by microsatellite markers and linkage analysis, but they were excluded based on the combination of haplotype analysis and two-point linkage analysis. The phenotype in this family is due to a mutation in another sutural cataract gene yet to be identified.

Amino Acid Sequence↗