Ralf Krahe | University of Texas M. D. Anderson Cancer Center (original) (raw)

Papers by Ralf Krahe

Genomics, Jul 1, 2000

Despite the central role of ␥-tubulin in the organization of the microtubule cytoskeleton, the ␥-... more Despite the central role of ␥-tubulin in the organization of the microtubule cytoskeleton, the ␥-tubulin gene family in humans has not been characterized. We now report the identification of a second expressed human ␥-tubulin gene (TUBG2) and a ␥-tubulin pseudogene (TUBG1P) in addition to the previously identified ␥-tubulin gene (TUBG1). Evidence from Southern hybridizations suggests that there are probably no additional ␥-tubulin sequences in the human genome. TUBG1 and TUBG2 are within 20 kb of each other in region q21 of chromosome 17, and TUBG1P is on chromosome 7. The proteins encoded by TUBG1 and TUBG2 share 97.3% amino acid identity, and the two genes are coexpressed in a variety of tissues. Previous studies of ␥-tubulin in human tissues and cell lines have been based on the tacit assumption that a single ␥-tubulin (the ␥-tubulin encoded by TUBG1) was present. While this assumption is not correct, the similarity of the products of TUBG1 and TUBG2 suggests that results of previous immunolocalization and immunoprecipitation studies in human cells and tissues are likely to be valid. In addition, any pharmacological agents that target one human ␥-tubulin are likely to target both.

PubMed, 1998

Mutations in the cystatin B (CSTB) gene underlie progressive myoclonus epilepsy of Unverricht-Lun... more Mutations in the cystatin B (CSTB) gene underlie progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) (Pennacchio et al., 1996). We previously described an unstable minisatellite expansion mutation in the putative promoter region of CSTB that accounts for the majority of EPM1 patients. Sequencing of a genomic lambda clone, generated from a Finnish EPM1 patient homozygous for an enlarged restriction fragment, revealed a 15- to 18-mer minisatellite repeat expansion (Virtaneva et al., 1997). Later, sequencing of plasmid clones generated from Swiss and French patients revealed a dodecamer repeat expansion (Lalioti et al., 1997a). By restriction enzyme analysis of our original patient clone and a clone generated from an Italian patient, we now show that the expansion is neither a 15-mer nor an 18-mer contrary to our initial results. Moreover, direct sequencing of the Finnish patient clone with Pfu exo polymerase confirmed that the expanded repeat is a dodecamer. Based on this finding and additional experiments, we suggest that the discrepancy between the two studies was due to errors caused by the combination of native Pfu polymerase and modified guanosine deaza-7-dGTP used in the PCR reaction.

European Journal of Neurology, Feb 1, 2005

Genes, Chromosomes and Cancer, 2005

Wilms tumor (WT) is genetically heterogeneous, and the one known WT gene, WT1 at 11p13, is altere... more Wilms tumor (WT) is genetically heterogeneous, and the one known WT gene, WT1 at 11p13, is altered in only a subset of WTs. Previous loss of heterozygosity (LOH) analyses have revealed the existence of additional putative WT genes at 11p15, 16q, and 1p, but these analyses examined only one or a handful of chromosomes or looked at LOH at only a few markers per chromosome. We conducted a genome-wide scan for LOH in WT by using 420 markers spaced at an average of 10 cM throughout the genome and analyzed the data for two genetically defined subsets of WTs: those with mutations in WT1 and those with no detectable WT1 alteration. Our findings indicated that the incidence of LOH throughout the genome was significantly lower in our group of WTs with WT1 mutations. In WT1-wild-type tumors, we observed the expected LOH at 11p, 16q, and 1p, and, in addition, we localized a previously unobserved region of LOH at 9q. Using additional 9q markers within this region of interest, we sublocalized the region of 9q LOH to the 12.2 Mb between D9S283 and a simple tandem repeat in BAC RP11-177I8, a region containing several potential tumor-suppressor genes. As a result, we have established for the first time that WT1-mutant and WT1-wild-type WTs differ significantly in their patterns of LOH throughout the genome, suggesting that the genomic regions showing LOH in WT1-wild-type tumors harbor genes whose expression is regulated by the pleiotropic effects of WT1. Our results implicate 9q22.2-q31.1 as a region containing such a gene.

Human Molecular Genetics, Aug 25, 2021

Myotonic dystrophy type 1 (DM1) is a complex disease with a wide spectrum of symptoms. The exact ... more Myotonic dystrophy type 1 (DM1) is a complex disease with a wide spectrum of symptoms. The exact relationship between mutant CTG repeat expansion size and clinical outcome remains unclear. DM1 congenital patients (CDM) inherit the largest expanded alleles, which are associated with abnormal and increased DNA methylation flanking the CTG repeat. However, DNA methylation at the DMPK locus remains understudied. Its relationship to DM1 clinical subtypes, expansion size and age-at-onset is not yet completely understood. Using pyrosequencing-based methylation analysis on 225 blood DNA samples from Costa Rican DM1 patients, we determined that the size of the estimated progenitor allele length (ePAL) is not only a good discriminator between CDM and non-CDM cases (with an estimated threshold at 653 CTG repeats), but also for all DM1 clinical subtypes. Secondly, increased methylation at both CTCF sites upstream and downstream of the expansion was almost exclusively present in CDM cases. Thirdly, levels of abnormal methylation were associated with clinical subtype, age and ePAL, with strong correlations between these variables. Fourthly, both ePAL and the intergenerational expansion size were significantly associated with methylation status. Finally, methylation status was associated with ePAL and maternal inheritance, with almost exclusively maternal transmission of CDM. In conclusion, increased DNA methylation at the CTCF sites flanking the DM1 expansion could be linked to ePAL, and both increased methylation and the ePAL could be considered biomarkers for the CDM phenotype.

Nature Genetics, Nov 1, 1999

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease with highly variable multi... more Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease with highly variable multisystemic manifestations. The mutation underlying DM is an unstable (CTG)n expansion in the 3´ UTR of the myotonic dystrophy protein kinase gene (DMPK). The pathophysiological mechanism(s) of the expanded (CTG)n repeat remains unclear. Various effects have been proposed, most recently a gain of function for mutant DMPK transcripts that results in a generalized RNA metabolism defect, mediated through one or several transacting proteins involved in RNA processing. This would in turn lead to the loss of function of multiple genes by qualitatively or quantitatively affecting post-transcriptional RNA processing, splicing or nuclear export of their transcripts. To test these hypotheses, we examined global mRNA expression changes between DM patients and normal controls by comprehensively and simultaneously profiling more than 6,800 human genes with oligo-based Genechip microarrays (Affymetrix). Total, nuclear and cytoplasmic RNA fractions of DM patient lymphoblastoid cell lines (four adult-onset, one congenital) as well as primary undifferentiated myoblasts and differentiated myotubes (one adult-onset, one congenital) were profiled. DM myoblasts in culture showed a reduced differentiation rate to myotubes and a tendency to dedifferentiate, suggesting a general block in or reprogramming of differentiation. Expression profiles of DM cell lines differed considerably from controls. Between the different DM cell lines profiled, many of the more than 6,800 genes assayed showed dysregulation. Moreover, comparison of nuclear and cytoplasmic fractions suggested a defect in the nuclear export of some processed transcripts. The number of genes dysregulated and the degree of dysregulation correlated with expansion size. Functions of the dysregulated genes were highly varied. In conclusion, DNA microarray expression profiling identified several novel DM phenotype effector candidate genes that may explain the complex pathogenesis of DM.

Neurology, Apr 24, 2019

Purpose of review Myotonic dystrophy type 2 (DM2) is a rare, progressive multisystem disease part... more Purpose of review Myotonic dystrophy type 2 (DM2) is a rare, progressive multisystem disease particularly affecting the skeletal muscle. A causal therapy is not yet available; however, prompt, appropriate symptomatic treatments are essential to limit disease-related complications. Evidence-based guidelines to assist medical practitioners in the care of DM2 patients do not exist. Recent findings The Myotonic Dystrophy Foundation (MDF) previously worked with an international group of 66 clinicians to develop consensusbased care recommendations for myotonic dystrophy type 1. Following a similar approach, the MDF recruited 15 international clinicians with long-standing experience in the care of DM2 patients to develop consensus-based care recommendations. The single text procedure was adopted. This process generated a 4-page Quick Reference Guide and a comprehensive 55-page document that provides care recommendations for DM2 patients.

Human Molecular Genetics

Myotonic dystrophy type 1 is a complex disease caused by a genetically unstable CTG repeat expans... more Myotonic dystrophy type 1 is a complex disease caused by a genetically unstable CTG repeat expansion in the 3′-untranslated region of the DMPK gene. Age-dependent, tissue-specific somatic instability has confounded genotype–phenotype associations, but growing evidence suggests that it also contributes directly toward disease progression. Using a well-characterized clinical cohort of DM1 patients from Costa Rica, we quantified somatic instability in blood, buccal cells, skin and skeletal muscle. Whilst skeletal muscle showed the largest expansions, modal allele lengths in skin were also very large and frequently exceeded 2000 CTG repeats. Similarly, the degree of somatic expansion in blood, muscle and skin were associated with each other. Notably, we found that the degree of somatic expansion in skin was highly predictive of that in skeletal muscle. More importantly, we established that individuals whose repeat expanded more rapidly than expected in one tissue (after correction for p...

Neuromuscular Disorders, 2003

Neuromuscular Disorders, 2017

High quality muscle biopsies are essential for imaging analyses. Detailed biopsy manuals were dev... more High quality muscle biopsies are essential for imaging analyses. Detailed biopsy manuals were developed for use by the surgical and laboratory teams at each site. With permission from Sarepta Therapeutics, manuals and training documents were modified to our specific clinical requirements. All sites were provided with detailed manuals and training sessions for relevant team members including surgeons and histotechnicians. One important training component involved practice of the freezing and shipping process using a beef sample. A pathologist from Flagship Bioscience then performed a quality control analysis of a beef cryosection from the shipped sample and confirmed successful site training prior to any patient biopsy. Biopsy samples from all UK and US sites were shipped to the Flagship Biosciences laboratory in the US for analysis. We have collected and assessed the quality of the biopsy samples from DMD boys in this way. In all cases, samples have been deemed of suitable quality for assay processing as confirmed by a pathologist's assessment of the stained cryosection. Using these biopsies and the recently developed validated imaging assays we expect to generate high quality data from all patients in the PhaseOut DMD study to evaluate target engagement and the effect of ezutromid on muscle fibre regeneration.

International Journal of Oncology, 2007

Cyclooxygenase-2 (COX-2) increases breast cancer cell invasion. Expression of various pro-angioge... more Cyclooxygenase-2 (COX-2) increases breast cancer cell invasion. Expression of various pro-angiogenic and pro-invasive factors has been correlated with high expression of COX-2. However, whether these factors are essential to COX-2-mediated breast cancer invasion, and the mechanisms by which COX-2 increases the expression of these factors are unknown. Our microarray results indicate that higher COX-2 expression was associated with increased levels of interleukin-8 (IL-8), a key factor in breast cancer invasion and metastasis. COX-2 overexpressing cells (MCF-7/COX-2), generated by transfecting COX-2-encoding plasmids into the poorly invasive MCF-7 breast cancer cells, were more invasive and produced higher IL-8 levels than the parental cells. To investigate the role of IL-8 in COX-2-mediated invasion, MCF-7 parental cells were incubated with IL-8. Exogenous IL-8 increased the invasiveness of MCF-7 cells. IL-8 is one pathway by which COX-2 mediates breast cancer invasion. Protein kinase A (PKA) and protein kinase C (PKC) are activated by COX-2 and are involved in IL-8 regulation. Inhibition of PKC, not PKA, decreased IL-8 production and invasion in MCF-7/COX-2 cells. Activation of PKC, not PKA, increased IL-8 production and invasion in MCF-7 cells. Thus, the invasive effects of COX-2 are mediated by PKC, not PKA. Activity of the urokinase-type plasminogen activator (uPA) was increased in MCF-7 cells by COX-2 overexpression or by the addition of a PKC activator or by IL-8. Inhibition of PKC decreased uPA activity in MCF-7/COX-2 cells. Furthermore, inhibition of uPA activity decreased the invasiveness of MCF-7/COX-2 cells, indicating that uPA was essential to COX-2-mediated invasion. Herein we demonstrate for the first time a detailed mechanism by which COX-2 increases breast cancer invasion: the PKC/IL-8/uPA pathway.

Neurology, 2004

To identify POLG mutations in patients with sensory ataxia and CNS features. The authors characte... more To identify POLG mutations in patients with sensory ataxia and CNS features. The authors characterized clinical, laboratory, and molecular genetic features in eight patients from five European families. The authors conducted sequencing of coding exons of POLG, C10orf2 (Twinkle), and ANT1 and analyzed muscle mitochondrial DNA (mtDNA), including Southern blot analysis and long-range PCR. Ataxia occurred in combination with various CNS features, including myoclonus, epilepsy, cognitive decline, nystagmus, dysarthria, thalamic and cerebellar white matter lesions on MRI, and neuronal loss in discrete gray nuclei on autopsy. Gastrointestinal dysmotility, weight loss, cardiomyopathy, and valproate-induced hepatotoxicity occurred less frequently. Two patients died without preceding signs of progressive external ophthalmoplegia. In muscle, typical findings of mitochondrial disease, such as ragged red fibers and Southern blot mtDNA abnormalities, were absent. POLG mutations were present in eight patients, including two isolated cases, and one Finnish and two unrelated Belgian families contained in total six patients. All POLG mutations were recessive, occurring in a homozygous state in seven patients and in a compound heterozygous state in one patient. The novel W748S mutation was identified in five patients from three unrelated families. The clinical spectrum of recessive POLG mutations is expanded by sensory ataxic neuropathy, combined with variable features of involvement of CNS and other organs. Progressive external ophthalmoplegia, myopathy, ragged red fibers, and Southern blot abnormalities of muscle mitochondrial DNA also are not mandatory features associated with POLG mutations.

American Journal of Human Genetics, Oct 1, 2003

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically an... more Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG) n expansion in the 3 untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG) n expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG) n expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG) n expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be ∼200-540 generations.

Journal of Osteoporosis

In previous study, we showed that nucleolar protein 66 (NO66) is a chromatin modifier and negativ... more In previous study, we showed that nucleolar protein 66 (NO66) is a chromatin modifier and negatively regulates Osterix activity as well as mesenchymal progenitor differentiation. Genetic ablation of the NO66 (RIOX1) gene in cells of the Prx1-expressing mesenchymal lineage leads to acceleration of osteochondrogenic differentiation and a larger skeleton in adult mice, whereas mesenchyme-specific overexpression of NO66 inhibits osteochondrogenesis resulting in dwarfism and osteopenia. However, the impact of NO66 overexpression in cells of the osteoblast lineage in vivo remains largely undefined. Here, we generated osteoblast-specific transgenic mice overexpressing a FLAG-tagged NO66 transgene driven by the 2.3 kB alpha-1type I collagen (Col1a1) promoter. We found that overexpression of NO66 in cells of the osteoblast lineage did not cause overt defects in developmental bones but led to osteoporosis in the long bones of adult mice. This includes decreased bone volume (BV), bone volume d...

Plot showing the average of three independent experiments for U251 glioblastoma cells in which ex... more Plot showing the average of three independent experiments for U251 glioblastoma cells in which exon 3 splicing was changed using targeted antisense morpholino oligonucleotide-treatment. The positions of five RefSeq entries representing are indicated. The inset shows representative RT-PCR results for exon 3 splicing following treatment with the control (MO-C) or antisense (MO-T) oligonucleotide. For data on RefSeq entries with significant values see Additional file . (B) Plot showing the genome-wide changes in expression and splicing observed in GBM compared to nontumor brain. For each Ref Seq entry, the values are derived from 24 GBM samples minus 12 nontumor brain samples. Notable RefSeq entries are labeled with their gene names. For data on RefSeq entries with significant values see Additional file . The theoretical values for a 5-fold (dashed line) and 10-fold (dotted line) change in exon inclusion are shown. For hybridization intensity maps of the highlighted genes see Additional file .<b>Copyright information:</b>Taken from "Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays"http://www.biomedcentral.com/1471-2164/9/216BMC Genomics 2008;9():216-216.Published online 12 May 2008PMCID:PMC2410136.

N-(4-Hydroxyphenyl)retinamide and nitric oxide pro-drugs exhibit apoptotic and anti-invasive effe... more N-(4-Hydroxyphenyl)retinamide and nitric oxide pro-drugs exhibit apoptotic and anti-invasive effects against bone metastatic breast cancer cells

PLOS ONE, 2019

Genotype-to-phenotype correlation studies in myotonic dystrophy type 1 (DM1) have been confounded... more Genotype-to-phenotype correlation studies in myotonic dystrophy type 1 (DM1) have been confounded by the age-dependent, tissue-specific and expansion-biased features of somatic mosaicism of the expanded CTG repeat. Previously, we showed that by controlling for the confounding effects of somatic instability to estimate the progenitor allele CTG length in blood DNA, age at onset correlations could be significantly improved. To determine the suitability of saliva DNA as a source for genotyping, we used small pool-PCR to perform a detailed quantitative study of the somatic mutational dynamics of the CTG repeat in saliva and blood DNA from 40 DM1 patients. Notably, the modal allele length in saliva was only moderately higher in saliva and not as large as previously observed in most other tissues. The lower boundary of the allele distribution was also slightly higher in saliva than it was in blood DNA. However, the progenitor allele length estimated in blood explained more of the variation in age at onset than that estimated from saliva. Interestingly, although the modal allele length was slightly higher in saliva, the overall degree of somatic variation was typically lower than in blood DNA, revealing new insights into the tissue-specific dynamics of somatic mosaicism. These data indicate that saliva constitutes an accessible, non-invasive and suitable DNA sample source for performing genetic studies in DM1.

Human Molecular Genetics, 2020

In myotonic dystrophy type 1 (DM1), somatic mosaicism of the (CTG)n repeat expansion is age-depen... more In myotonic dystrophy type 1 (DM1), somatic mosaicism of the (CTG)n repeat expansion is age-dependent, tissue-specific and expansion-biased. These features contribute toward variation in disease severity and confound genotype-to-phenotype analyses. To investigate how the (CTG)n repeat expansion changes over time, we collected three longitudinal blood DNA samples separated by 8–15 years and used small pool and single-molecule PCR in 43 DM1 patients. We used the lower boundary of the allele length distribution as the best estimate for the inherited progenitor allele length (ePAL), which is itself the best predictor of disease severity. Although in most patients the lower boundary of the allele length distribution was conserved over time, in many this estimate also increased with age, suggesting samples for research studies and clinical trials should be obtained as early as possible. As expected, the modal allele length increased over time, driven primarily by ePAL, age-at-sampling and...

Duodecim lääketieteellinen aikakauskirja, 2003