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Jochen Graw

Publications and source records attributed to Jochen Graw.

At least 19 recordsLinked to original sources
PubMed2006

Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans.

MENX is a recessive multiple endocrine neoplasia-like syndrome in the rat. The tumor spectrum in MENX overlaps those of human multiple endocrine neoplasia (MEN) types 1 and 2. We mapped the MenX locus to the distal part of rat chromosome 4, excluding the homologs of the genes responsible for the MEN syndromes (RET and MEN1) and syndromes with an endocrine tumor component (VHL and NF1). We report the fine mapping of the disease locus and the identification of a homozygous frameshift mutation in Cdkn1b, encoding the cyclin-dependent kinase inhibitor p27(Kip1). As a consequence of the mutation, MENX-affected rats show dramatic reduction in p27(Kip1) protein. We have identified a germ-line nonsense mutation in the human CDKN1B gene in a MEN1 mutation-negative patient presenting with pituitary and parathyroid tumors. Expanded pedigree analysis shows that the mutation is associated with the development of an MEN1-like phenotype in multiple generations. Our findings demonstrate that germ-line mutations in p27(Kip1) can predispose to the development of multiple endocrine tumors in both rats and humans.

Amino Acid Sequence↗
PubMed2006

CRYBA4, a novel human cataract gene, is also involved in microphthalmia.

Genetic analysis of a large Indian family with an autosomal dominant cataract phenotype allowed us to identify a novel cataract gene, CRYBA4. After a genomewide screen, linkage analysis identified a maximum LOD score of 3.20 (recombination fraction [theta] 0.001) with marker D22S1167 of the beta -crystallin gene cluster on chromosome 22. To date, CRYBA4 was the only gene in this cluster not associated with either human or murine cataracts. A pathogenic mutation was identified in exon 4 that segregated with the disease status. The c.317T-->C sequence change is predicted to replace the highly conserved hydrophobic amino acid phenylalanine94 with the hydrophilic amino acid serine. Modeling suggests that this substitution would significantly reduce the intrinsic stability of the crystalline monomer, which would impair its ability to form the association modes critical for lens transparency. Considering that CRYBA4 associates with CRYBB2 and that the latter protein has been implicated in microphthalmia, mutational analysis of CRYBA4 was performed in 32 patients affected with microphthalmia (small eye). We identified a c.242T-->C (Leu69Pro) sequence change in exon 4 in one patient, which is predicted here to disrupt the beta -sheet structure in CRYBA4. Protein folding would consequently be impaired, most probably leading to a structure with reduced stability in the mutant. This is the first report linking mutations in CRYBA4 to cataractogenesis and microphthalmia.

Adult↗
PubMed2006

Variations of eye size parameters among different strains of mice.

In the mouse, only a few genes have been definitively associated with a small-eye phenotype; the paired-box gene Pax6 and the gene coding for the microphthalmia-associated transcription factor (Mitf). Mutant alleles were recovered by crude phenotype screens and their effects on eye size are relatively large. This feature points to a bias during screening for eye-size mutants, selecting preferentially more severe phenotypes. An unbiased method determining eye-size parameters in an observer-independent, quantitative manner is expected to pick up variations in other genes, which will be confirmed as pathologic mutations in confirmation crosses. The present study used optical low coherent interferometry (OLCI) to compare the axial eye length, the cornea and lens thicknesses, and the anterior chamber depth in four common wild-type, laboratory inbred strains (C57BL/6J, C3HeB/FeJ, 129S2/SvPasCrl, and BALB/cByJ) between 4 and 15 weeks of age. There were no differences between left and right eyes; differences between the size parameters of males and females have been observed only in a few cases. An optimal screening age for OLCI measurements was defined as 11 weeks of age. At this age, we checked two other inbred strains (AKR/J and DBA/2NCrl) as well as CD-1 outbred mice. CD-1 mice have the largest axial length. The most impressive differences among inbred strains were, first, the anterior chamber depth, where the DBA mice have significantly lower values than the other strains. Second, the cornea in C3H mice is approximately 20% thicker than in the other inbred strains. Finally, wild-type intervals (mean +/- 3 SD) for axial length, anterior chamber depth, and cornea and lens thicknesses were calculated allowing a quick identification of pathologic outliers.

Animals↗
PubMed2006

Identification of a novel, putative cataract-causing allele in CRYAA (G98R) in an Indian family.

PURPOSE: The aim of the present study was to investigate the molecular basis underlying a nonsyndromic presenile autosomal dominant cataract in a three-generation pedigree. The phenotype was progressive from a peripheral ring-like opacity to a total cataract with advancing age from teenage to adulthood. The visual impairment started as problem in distant vision at the age of 16 years, to diminishing vision by the age of 24. METHODS: Clinical interventions included complete ophthalmological examination, a collection of case history, and pedigree details. Blood samples were collected from available family members irrespective of their clinical status. A functional candidate gene approach was employed for PCR screening and sequencing of the exons and their flanking regions of CRYGC, CRYGD, and CRYAA genes. For structural consequences of the mutated alphaA-crystallin we used the bioinformatics tool of the ExPASy server. RESULTS: Sequence analysis of CRYGC and CRYGD genes excluded possible causative mutations but identified known polymorphisms. Sequencing of the exons of the CRYAA gene identified a sequence variation in exon 2 (292 G->A) with a substitution of Gly to Arg at position 98. All three affected members revealed this change but it was not observed in the unaffected father or sister. The putative mutation obliterated a restriction site for the enzyme BstDSI. The same was checked in controls representing the general population of the same ethnicity (n=30) and of randomly selected DNA samples from ophthalmologically normal individuals from the population-based KORA S4 study (n=96). Moreover, the Gly at position 98 is highly conserved throughout the animal kingdom. For the mutant protein, the isoelectric point was raised from pH 5.77 to 5.96. Moreover, an extended alpha-helical structure is predicted in this region. CONCLUSIONS: The G98R mutation segregates only in affected family members and is not seen in representative controls. It represents very likely the fourth dominant cataract-causing allele in CRYAA. In all reported alleles the basic amino acid Arg is involved, suggesting the major importance of the net charge of the alphaA-crystallin for functional integrity in the lens.

Adult↗
PubMed2006

Congenital cataract and macular hypoplasia in humans associated with a de novo mutation in CRYAA and compound heterozygous mutations in P.

BACKGROUND: An isolated form of congenital cataract associated with macular hypoplasia and a generally hypopigmented fundus in infancy was observed in a German family. To test the hypothesis that a de-novo mutation had occurred in one of the parental germ lines, a functional candidate gene approach was applied. METHODS: The family was carefully examined by a senior paediatric ophthalmologist according to routine procedures (slit lamp, funduscopy, ERG). Blood was taken from the proband and his parents, genomic DNA was isolated and some candidate genes for cataract (CRYAA, CRYBB2, GJA8) or macular hypoplasia (OA1, P) or both (PAX6) were analyzed. RESULTS: The proband showed bilateral cataracts at the age of 4 months; the fundus appeared pale, the optic disc grayish, and macular reflexes were absent. After cataract surgery, the nystagmus persisted, and a control ERG at age 9 years showed essentially normal scotopic and photopic wave forms. An infectious aetiology as well as galactosemia were excluded. However, a heterozygous mutation was found in the proband in exon 1 of CRYAA (62 C-->T), which leads to an exchange from Arg to Leu at amino acid position 21 (R21L). This sequence alteration was not found in the parents and in 96 randomly selected DNA samples from ophthalmologically normal individuals of the KORA S4 study population. In addition, two heterozygous mutations in P were identified (R419Q and A481T); one of both was present in each of the unaffected parents. CONCLUSION: Based upon the unique finding of the mutation and the expression of CRYAA in the lens, this R21L mutation in the CRYAA is considered to be causative for the dominant cataract phenotype. Moreover, the macular hypoplasia has to be considered a concerted interaction with compound heterozygous mutations in the P gene manifesting a mild form of oculocutaneous albinism. Nevertheless, this combination is rare and future studies will focus on identifying similar phenotypes.

Cataract↗
PubMed2005

Lack of F8 mRNA: a novel mechanism leading to hemophilia A.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8 gene was detected. Using 2 common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription-polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARA locus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, which points to a novel mechanism leading to HA.

Adolescent↗
PubMed2005

Betacellulin overexpression in transgenic mice causes disproportionate growth, pulmonary hemorrhage syndrome, and complex eye pathology.

The EGF family comprises a network of ligands and receptors that regulate proper development and elicit diverse functions in physiology and pathology. Betacellulin (BTC) is a rather poorly characterized member of the EGF family whose in vivo effects have been linked mainly to endocrine pancreas, intestine, and mammary gland function. In vitro studies revealed that this growth factor is a potent mitogen for diverse cell types and suggested unique receptor-binding properties. Genetic ablation of BTC in mice yielded a mild phenotype, probably because of opportunistic compensation by other EGF receptor ligands. To study the biological capabilities of BTC in vivo, we generated transgenic mice overexpressing BTC ubiquitously, with highest expression levels in heart, lung, brain, and pancreas. Mice overexpressing BTC exhibit high early postnatal mortality, reduced body weight gain, and impaired longitudinal growth. In addition, a variety of pathological alterations were observed. Cataract and abnormally shaped retinal layers as well as bone alterations leading to a dome-shaped, round head form were hallmarks of BTC transgenic mice. The most important finding and the cause of reduced life expectancy of BTC transgenic mice were severe alterations of the lung. Pulmonary pathology was primarily characterized by alveolar hemorrhage, thickening of the alveolar septa, intraalveolar accumulation of hemosiderin-containing macrophages, and nodular pulmonary remodeling. Thus, our model uncovers multiple consequences of BTC overexpression in vivo. These transgenic mice provide a useful model for examining the effects of BTC excess on different organs.

Animals↗
PubMed2005

Agonistic and antagonistic action of AP2, Msx2, Pax6, Prox1 AND Six3 in the regulation of Sox2 expression.

Sox2 transcription factor is expressed in neural tissues and sensory epithelia from the early stages of development. Particularly, it is known to activate crystallin gene expression and to be involved in differentiation of lens and neural tissues. However, its place in the signaling cascade is not well understood. Here, we report about the response of its promoter to the presence of other transcription factors, AP2alpha, Msx2, Pax6, Prox1 and Six3, in a transient reporter gene assay using HEK293 cells as recipient cells. Taking our data together, AP2, Pax6 and PROX1 can activate the Sox2 promoter. Msx2 has an inhibitory effect, whereas Six3 does not affect the Sox2 promoter. These data indicate a common activating cascade at least for AP2, Pax6, Prox1 and Sox2.

Blotting, Western↗
PubMed2005

An ENU-induced mutation in Rs1h causes disruption of retinal structure and function.

PURPOSE: The 44TNJ mutant mouse was generated by the Tennessee Mouse Genome Consortium (TMGC) using an ENU-based mutagenesis screen to produce recessive mutations that affect the eye and brain. Herein we present its retinal phenotype and genetic basis. METHODS: Fourth generation offspring (G4) and confirmed mutants were examined using slit lamp biomicroscopy, funduscopy, histology, immunohistochemistry, and electroretinography (ERG). 44TNJ mutant mice were crossed to C3BLiA or DBA/2 mice for chromosomal mapping purposes. Linkage analysis by PCR-based microsatellite marker genotyping was used to identify the disease locus. The Rs1h cDNA and its genomic DNA were sequenced directly. RESULTS: The 44TNJ pedigree was the first mutant pedigree identified by the ocular phenotyping domain of the TMGC. Examination of the fundus revealed numerous small and homogeneous intraretinal microflecks in the peripapillary region, which became courser and more irregular in the periphery. Males were typically more affected than females. Histology and immunohistochemistry revealed a disruption of the lamination of the retina, particularly at both margins of the outer nuclear layer, along with reduced calbindin immunostaining. ERG analyses revealed reduced amplitudes of both a-waves and b-waves. Linkage analysis mapped the 44TNJ mutation to the X chromosome close to the marker DXMit117. Sequence analysis of the positional candidate gene Rs1h revealed a T->C exchange at the second base of intron 2 of the Rs1h gene. CONCLUSIONS: We have generated and characterized a mutant mouse line that was produced using ENU-based mutagenesis. The 44TNJ pedigree manifests with photoreceptor dysfunction and concurrent structural and functional aberrations at the post-receptoral level. Genetic analysis revealed a mutation in Rs1h, making this the first murine model of X-linked retinoschisis in which the gene is expressed.

Alkylating Agents↗
PubMed2005

Mouse mutants as models for congenital retinal disorders.

Animal models provide a valuable tool for investigating the genetic basis and the pathophysiology of human diseases, and to evaluate therapeutic treatments. To study congenital retinal disorders, mouse mutants have become the most important model organism. Here we review some mouse models, which are related to hereditary disorders (mostly congenital) including retinitis pigmentosa, Leber's congenital amaurosis, macular disorders and optic atrophy.

Animals↗
PubMed2005

Haemophilia A: from mutation analysis to new therapies.

Haemophilia is caused by hundreds of different mutations and manifests itself in clinical conditions of varying severity. Despite being inherited in monogenic form, the clinical features of haemophilia can be influenced by other genetic factors, thereby confounding the boundary between monogenic and multifactorial disease. Unlike sufferers of other genetic diseases, haemophiliacs can be treated successfully by intravenous substitution of coagulation factors. Haemophilia is also the most attractive model for developing gene-therapy protocols, as the normal life expectancy of haemophiliacs allows the side effects of gene therapy, as well as its efficiency, to be monitored over long periods.

DNA Mutational Analysis↗
PubMed2005

Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters.

PURPOSE: To characterize three new mouse small-eye mutants detected during ethylnitrosourea mutagenesis programs. METHODS: Three new mouse small-eye mutants were morphologically characterized, particularly by in situ hybridization. The mutations were mapped, and the candidate gene was sequenced. The relative amount of Pax6-specific mRNA was determined by real-time PCR. Reporter gene analysis used Crygf and Six3 promoter fragments in front of a luciferase gene and HEK293 cells as recipients. RESULTS: The new mutations--ADD4802, Aey11, and Aey18--were mapped to chromosome 2; causative mutations have been characterized in Pax6 (Aey11: C-->T substitution in exon 8, creating a stop codon just in front of the homeobox; ADD4802: G-->A substitution at the beginning of intron 8 changes splicing and leads to an altered open reading frame and then to a premature stop codon; Aey18: G-->A exchange in the last base of intron 5a leads also to a splice defect, skipping exons 5a and 6). Real-time PCR indicated nonsense-mediated decay in Pax6Aey11 and Pax6Aey18 mutants but not in Pax6ADD4802. This result is supported by the functional analysis of corresponding expression constructs in cell culture, where the Aey11 and Aey18 alleles did not show a stimulation of the Six3 promotor or an inhibition of the Crygf promoter (as wild-type constructs do). However, the Pax6ADD4802 allele stimulated both promoters. CONCLUSIONS: Together with functional analysis in a reporter gene assay and immunohistochemistry using Pax6 antibodies, it is suggested that the Pax6Aey11 and Pax6Aey18 alleles act through a loss of function, whereas ADD4802 represents a gain-of-function allele.

Alleles↗
PubMed2004

Molecular dissection of Pax6 function: the specific roles of the paired domain and homeodomain in brain development.

The transcription factor Pax6 plays a key role during development of various organs, including the brain where it affects cell fate, cell proliferation and patterning. To understand how Pax6 coordinates these diverse effects at the molecular level, we examined the role of distinct DNA-binding domains of Pax6, the homeodomain (HD), the paired domain (PD) and its splice variant (5a), using loss- and gain-of-function approaches. Here we show that the PD is necessary for the regulation of neurogenesis, cell proliferation and patterning effects of Pax6, since these aspects are severely affected in the developing forebrain of the Pax6Aey18 mice with a deletion in the PD but intact homeo- and transactivation domains. In contrast, a mutation of the HD lacking DNA-binding (Pax64Neu) resulted in only subtle defects of forebrain development. We further demonstrate distinct roles of the two splice variants of the PD. Retrovirally mediated overexpression of Pax6 containing exon 5a inhibited cell proliferation without affecting cell fate, while Pax6 containing the canonical form of the PD lacking exon 5a affected simultaneously cell fate and proliferation. These results therefore demonstrate a key role of the PD in brain development and implicate splicing as a pivotal factor regulating the potent neurogenic role of Pax6.

Alternative Splicing↗
PubMed2004

Mapping of a novel MEN-like syndrome locus to rat chromosome 4.

Multiple endocrine neoplasia-like syndrome (MENX) is a hereditary cancer syndrome in the rat characterized by inborn cataract and multiple tumors affecting the neuroendocrine system developed within the first year of life. The spectrum of affected organs is intermediate between MEN type 1 (MEN1) and MEN type 2 (MEN2) syndromes in human, but, in contrast to them, MENX is inherited in a recessive fashion. Here we report the mapping of the MENX locus to rat Chromosome (Chr) 4 by a genome-wide linkage analysis. This analysis was done in 41 animals obtained from a (Wistar/Nhg x SDwe) x SDwe interstrain backcross, where SDwe (Sprague-Dawley white eye) indicates the affected animals. The MENX disease locus was ultimately mapped to a approximately 22-cM interval on Chr 4 that includes the rat homolog of the human RET proto-oncogene. As activating point mutations of RET are known to be responsible for MEN2 in human, we analyzed several markers located in the proximity of Ret for linkage to the disease phenotype. Our data exclude Ret involvement in MENX and establish that a second gene, playing a role in endocrine tumor formation, lies within the distal part of rat Chr 4. Although heritable human endocrine tumors are quite rare, sporadic tumors of MEN-affected tissues occur at a much higher frequency, and their pathogenesis is poorly understood. The identification of the MENX gene should contribute to our understanding of the genetic mechanisms of neuroendocrine tissue tumorigenesis and may assist in developing new and more appropriate therapeutic strategies for these diseases.

Animals↗
PubMed2004

Electroretinography as a screening method for mutations causing retinal dysfunction in mice.

PURPOSE: To detect mice with hereditary retinal impairment, a high-throughput electroretinography (ERG) screening system was established. METHOD: Mice from eight different strains without known retinal disorders (102, 129/SvJ, AKR, C57BL/6J, C57BL/6JIco, CBA/CaJ, and DBA/2NCrlBR) and one control strain with retinal degeneration (C3HeB/FeJ) were fixed on a specially constructed sled, ERG electrodes were placed on the cornea, and mice were moved into a Ganzfeld stimulator. From a luminance range of 0.0125 to 500 cd-s/m(2) in a pretest series two levels (5 and 125 cd-s/m(2)) were chosen to shorten examination times. The root mean square (RMS) of the ERG-recording was analyzed to detect animals with abnormal retinal function. ERG responses of the left and right eyes were compared in amplitudes and implicit times of the a- and b-waves. Statistical analysis of the latter parameters was performed in all wild-type animals. Histology was performed on selected mice. RESULTS: ERG recordings of individual animals for the left and right eye revealed good agreement in amplitudes and implicit times of the a- and b-waves (P < 0.05). Comparison of these parameters among the wild-type strains showed several differences. Evaluation of the RMS revealed, in addition to the C3HeB/FeJ mice, a subgroup of mice within the 129/SvJ strain with abnormal retinal function. Molecular analysis of these mice demonstrated the presence of the same retroviral insertion in the Pde6b gene, which is causative of the Pde6b(rd1) allele carried in C3HeB/FeJ mice. Histologic analysis demonstrated good correlation between retinal electrophysiology and morphology. CONCLUSIONS: The present results demonstrate the feasibility of ERG for screening a large number of mice to detect animals with functional retinal impairment.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

Genetic and allelic heterogeneity of Cryg mutations in eight distinct forms of dominant cataract in the mouse.

PURPOSE: The purpose of this study was the characterization of eight new dominant cataract mutations. METHODS: Lenses of mutant mice were described morphologically and histologically. Each mutation was mapped by linkage studies. The candidate genes (the Cryg gene cluster and the closely linked Cryba2 gene) were sequenced. RESULTS: Molecular analysis confirmed all mutations in Cryg genes. Five mutations lead to amino acid exchanges, two are due to premature stop codons, and one is a 10-bp deletion in the Cryge gene. Morphologically, mutant carriers expressed nonsyndromic cataracts, ranging from diffuse lenticular opacities (Crygd(ENU910) and Cryge(ENU449)), to dense nuclear and subcortical opacity (Crygd(K10), Crygc(MNU8), Cryge(Z2), Crygd(ENU4011), and Cryge(ADD15306)), to dense nuclear opacity and ruptured lenses (Cryga(ENU469)). Results of histologic analyses correlate well with the severity of lens opacity, ranging from alterations in the process of secondary fiber nucleus degradation to lens vacuoles, fiber degeneration, and disruption of the lens capsule. CONCLUSIONS: In total, 20 mutations have been described that affect the Cryg gene cluster: Nine mutations affect the Cryge gene, but only one affects the Crygb or Crygf genes. No mutation was observed in the closely linked Cryba2. Two mutations occur at the same site in the Crygd and Cryge genes (Leu45-->Pro). The unequal distribution of mutations suggests hot spots in the Cryg genes. The overall high number of mutations in these genes demonstrates their central role in the maintenance of lens transparency.

Alleles↗