Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis.

Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.

New insights in the molecular pathogenesis of the maternally inherited diabetes and deafness syndrome.

The 3243A>G mutation in mitochondrial DNA (mtDNA) is a genetic variant that is associated with a high risk of developing diabetes during life. Enhanced aging of pancreatic beta-cells, a reduced capacity of these cells to synthesize large amounts of insulin,and a resetting of the ATP/ADP-regulated K-channel seem to be the pathogenic factors involved.

Real-time monitoring of rolling-circle amplification using a modified molecular beacon design.

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.

Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles.

A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.

Mitochondrial diabetes: molecular mechanisms and clinical presentation.

Mutations in mitochondrial DNA (mtDNA) associate with various disease states. A few mtDNA mutations strongly associate with diabetes, with the most common mutation being the A3243G mutation in the mitochondrial DNA-encoded tRNA(Leu,UUR) gene. This article describes clinical characteristics of mitochondrial diabetes and its molecular diagnosis. Furthermore, it outlines recent developments in the pathophysiological and molecular mechanisms leading to a diabetic state. A gradual development of pancreatic beta-cell dysfunction upon aging, rather than insulin resistance, is the main mechanism in developing glucose intolerance. Carriers of the A3243G mutation show during a hyperglycemic clamp at 10 mmol/l glucose a marked reduction in first- and second-phase insulin secretion compared with noncarriers. The molecular mechanism by which the A3243G mutation affects insulin secretion may involve an attenuation of cytosolic ADP/ATP levels leading to a resetting of the glucose sensor in the pancreatic beta-cell, such as in maturity-onset diabetes of the young (MODY)-2 patients with mutations in glucokinase. Unlike in MODY2, which is a nonprogressive form of diabetes, mitochondrial diabetes does show a pronounced age-dependent deterioration of pancreatic function indicating involvement of additional processes. Furthermore, one would expect that all mtDNA mutations that affect ATP synthesis lead to diabetes. This is in contrast to clinical observations. The origin of the age-dependent deterioration of pancreatic function in carriers of the A3243G mutation and the contribution of ATP and other mitochondrion-derived factors such as reactive oxygen species to the development of diabetes is discussed.

A multiplexed and miniaturized serological tuberculosis assay identifies antigens that discriminate maximally between TB and non-TB sera.

We have developed a multiplexed and miniaturized TB serological assay with the aim of identifying (combinations of) antigens that maximally discriminate between TB and non-TB patients. It features a microarray accommodating 54 TB antigens, less than 1 microl serum consumption and an indirect immunofluorescence detection protocol. With a panel of 20 TB and 80 non-TB sera we ranked combinations of TB antigens with respect to sensitivity and specificity of TB detection by means of logistic step-forward regression analysis. The highest-ranking TB antigen combination had an area-under-the-curve of the receiver-operator-characteristics (ROC) of 0.95. We also identified an antigen that on its own provided good specificity and sensitivity of TB detection (Ara6-BSA; area-under-the-ROC curve: 0.90). These area-under-the-ROC curve values are exceptionally high for a serological TB assay. We conclude that TB antigen microarrays permit rapid identification of TB antigens that, either alone or in combination, discriminate maximally between TB and non-TB patients and that such identification provides an excellent starting point for developing point-of-care diagnostic assays.

Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation.

The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.

Localization and regulation of the growth hormone receptor and growth hormone-binding protein in the rat growth plate.

Growth hormone (GH) has direct effects on the growth plate to stimulate longitudinal growth, but it is not clear which chondrocyte populations GH acts on. The dual effector theory suggests that GH would act primarily on the "stem cells." However, staining with a GH receptor (GHR) antibody is found in all layers of the growth plate in rabbits and humans. We now have investigated the localization and regulation of GHR and the related GH binding protein (GHBP) in the rat growth plate using a sensitive immunohistochemical method involving tyramide signal amplification (TSA) and antibodies specific for GHR or GHBP. Both GHR and GHBP were shown in the germinal and proliferative chondrocytes, but most clearly in early maturing chondrocytes at the interface between proliferative and hypertrophic cells. Staining for GHR and GHBP was located in both the cytoplasm and the nucleus. Expression of GHR mRNA and GHBP mRNA in the growth plate was confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Immunohistochemical staining for GHR and GHBP decreased with age; in 12-week-old normal rats, only the early maturing chondrocytes were stained. In GH-deficient dwarf rats, staining seemed less than in normal rats, and in hypophysectomized (Hx) rats, staining for GHBP was clearly reduced. Treatment of Hx rats with thyroid hormones (T3 + T4), via subcutaneously (sc) implanted osmotic minipumps, induced little growth and induced a small layer of GHR-positive and GHBP-positive early maturing chondrocytes. Treatment with GH and thyroid hormones (TH) resulted in greater growth and a broader layer of GHR-positive and GHBP-positive cells, indistinguishable from normal rats. In contrast, dexamethasone treatment of normal rats inhibited their growth and reduced GHR and GHBP staining in the growth plate. These results show that GHR and GHBP in the growth plate are under hormonal control. The localization of GHR/GHBP suggests that in addition to actions on germinal and proliferative cells in young rats, GH also has effects on early maturing chondrocytes and may be involved in their differentiation to a fully hypertrophic chondrocyte.

Connexin 26 mutations in cases of sensorineural deafness in eastern Austria.

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G-->T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.

Comprehensive analysis of human subtelomeres with combined binary ratio labelling fluorescence in situ hybridisation.

Cryptic subtelomeric chromosome rearrangements play an important role in the aetiology of mental retardation, congenital anomalies, miscarriages and neoplasia. To facilitate a comprehensive molecular-cytogenetic analysis of these extremely gene-rich and mutation-prone chromosome regions, novel multicolour fluorescence in situ hybridisation (FISH) techniques are being developed. As yet, subtelomeric FISH methods have either had limited multiplicities, making it necessary to perform many hybridisations per patient, or a limited scope of analysable chromosome mutation types, thus not detecting some aberration types such as pericentric inversions or very small aberrations. COBRA (COmbined Binary RAtio) labelling is a generic multicolour FISH technique that combines ratio and combinatorial labelling to attain especially high multiplicities with few fluorochromes. The Subtelomere COBRA FISH method ("S-COBRA FISH") described here detects efficiently all 41 BAC and PAC FISH probes necessary for a complete subtelomere screening in only two hybridisations. It was applied to the analysis of 10 cases with known and partially known aberrations and successfully detected balanced and unbalanced translocations, deletions and an unbalanced pericentric inversion in a mosaic situation. The ability of S-COBRA FISH to efficiently detect all types of balanced and unbalanced subtelomeric chromosome aberrations makes it the most comprehensive diagnostic procedure for human subtelomeric chromosome regions described to date.

Array comparative genomic hybridization with cyanin cis-platinum-labeled DNAs.

Fluorescent cis-platinum compounds that react with the N7 atom of guanine are useful for labeling nucleic acids influorescence hybridization applications. Here we report that cyanin (CyN) cis-platinum labeling of DNA samples for array comparative genomic hybridizations (arrayCGH) can be achieved reproducibly and reliably. We demonstrate that degrees of labeling of approximately 1% of all nucleotides in test and reference DNA samples with CyN3- and CyN5-cis-platinum produces arrayCGH signal-to-background ratios ranging from 30 to 40. The arrayCGH results achieved during analyses of mouse and human tumor samples were comparable to those achieved using enzymatic labeling. Thus, we conclude that Cy-cis-platinum labeling is an alternative to enzymatic labeling for arrayCGH.

A novel t(6;14)(q25-q27;q32) in acute myelocytic leukemia involves the BCL11B gene.

Cytogenetic studies in a patient with acute myelocytic leukemia (AML) revealed as the sole karyotypic alteration a half-cryptic rearrangement, identified with 48-color combined binary ratio-labeled fluorescence in situ hybridization (pq-COBRA-FISH) as a reciprocal t(6;14)(q?;q?). The breakpoints were later assigned on the basis of G-banding to t(6;14)(q25-q26;q32). FISH experiments using genomic probes showed that the breakpoint on 14q32.2 was within bacterial artificial chromosome RP11-782I5 and revealed BCL11B as the only candidate gene in the region. BCL11B is a homolog to BCL11A (2p13), a highly conserved gene implicated in mouse and human leukemias. To our knowledge, this is the first report implicating BCL11B in hematological malignancies. Because of lack of material, the translocation partner remains unknown.

Positioning of cervical carcinoma and Burkitt lymphoma translocation breakpoints with respect to the human papillomavirus integration cluster in FRA8C at 8q24.13.

Molecular cytogenetic analysis frequently shows human papillomavirus (HPV) integration near translocation breakpoints in cervical cancer cells. We have recently described a cluster of HPV18 integrations in the distal end of the common fragile site FRA8C at 8q24 in primary cervical carcinoma samples. Chromosome band 8q24 contains the MYC gene (alias c-MYC), FRA8C, and FRA8D. The MYC gene is frequently deregulated--usually by translocation or amplification--in various tumor types. In the present study, we performed a molecular cytogenetic analysis of HPV18 integration patterns and the 8q24 translocation in a primary cervical carcinoma and in HeLa cells using combined binary ratio-fluorescence in situ hybridization. Our aim was to determine how the chromosomal breaks involved in these events relate physically to the MYC gene; whether they map to the FRA8C site, the FRA8D site, or both; and how they correlate with the occurrence of DNA flexibility domains. The 8q24 translocation breakpoints mapped between stretches of integrated HPV18 sequences in the distal end of FRA8C. This region contained DNA helix flexibility clusters, several of which mapped in the vicinity of HPV integration sites and translocation breakpoints in cervical carcinomas. DNA helix flexibility clusters were also found near known MYC translocation breakpoints in Burkitt lymphomas (BL), but most BL breakpoints mapped clearly outside FRA8C. Our data revealed that FRA8C is involved in HPV integration and chromosomal translocations in cervical carcinoma; however, this fragile site is not involved in classical MYC translocations in most BLs. In the context of the familial nature of cervical cancer, FRA8C may be considered a candidate susceptibility region for cervical carcinoma.

Simultaneous mapping of human papillomavirus integration sites and molecular karyotyping in short-term cultures of cervical carcinomas by using 49-color combined binary ratio labeling fluorescence in situ hybridization.

Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes' shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21 through 23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures.

Non-random mtDNA segregation patterns indicate a metastable heteroplasmic segregation unit in m.3243A>G cybrid cells.

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.

The A3243G tRNALeu(UUR) mutation induces mitochondrial dysfunction and variable disease expression without dominant negative acting translational defects in complex IV subunits at UUR codons.

Mutations in the mitochondrial tRNA(Leu(UUR)) gene are associated with a large variety of human diseases through a largely undisclosed mechanism. The A3243G tRNA(Leu(UUR)) mutation leads to reduction of mitochondrial DNA (mtDNA)-encoded proteins and oxidative phosphorylation activity even when the cells are competent in mitochondrial translation. These two aspects led to the suggestion that a dominant negative factor may underlie the diversity of disease expression. Here we test the hypothesis that A3243G tRNA(Leu(UUR)) generates such a dominant negative gain-of-function defect through misincorporation of amino acids at UUR codons of mtDNA-encoded proteins. Using an anti-complex IV immunocapture technique and mass spectrometry, we show that the mtDNA-encoded cytochrome c oxidase I (COX I) and COX II exist exclusively with the correct amino acid sequences in A3243G cells in a misassembled complex IV. A dominant negative component therefore cannot account for disease phenotype, leaving tissue-specific accumulation by mtDNA segregation as the most likely cause of variable mitochondrial disease expression.

Array-based comparative genomic hybridization analysis reveals recurrent chromosomal alterations and prognostic parameters in primary cutaneous large B-cell lymphoma.

PURPOSE: To evaluate the clinical relevance of genomic aberrations in primary cutaneous large B-cell lymphoma (PCLBCL). PATIENTS AND METHODS: Skin biopsy samples of 31 patients with a PCLBCL classified as either primary cutaneous follicle center lymphoma (PCFCL; n = 19) or PCLBCL, leg type (n = 12), according to the WHO-European Organisation for Research and Treatment of Cancer (EORTC) classification, were investigated using array-based comparative genomic hybridization, fluorescence in situ hybridization (FISH), and examination of promoter hypermethylation. RESULTS: The most recurrent alterations in PCFCL were high-level DNA amplifications at 2p16.1 (63%) and deletion of chromosome 14q32.33 (68%). FISH analysis confirmed c-REL amplification in patients with gains at 2p16.1. In PCLBCL, leg type, most prominent aberrations were a high-level DNA amplification of 18q21.31-q21.33 (67%), including the BCL-2 and MALT1 genes as confirmed by FISH, and deletions of a small region within 9p21.3 containing the CDKN2A, CDKN2B, and NSG-x genes. Homozygous deletion of 9p21.3 was detected in five of 12 patients with PCLBCL, leg type, but in zero of 19 patients with PCFCL. Complete methylation of the promoter region of the CDKN2A gene was demonstrated in one PCLBCL, leg type, patient with hemizygous deletion, in one patient without deletion, but in zero of 19 patients with PCFCL. Seven of seven PCLBCL, leg type, patients with deletion of 9p21.3 and/or complete methylation of CDKN2A died as a result of their lymphoma. CONCLUSION: Our results demonstrate prominent differences in chromosomal alterations between PCFCL and PCLBCL, leg type, that support their classification as separate entities within the WHO-EORTC scheme. Inactivation of CDKN2A by either deletion or methylation of its promoter could be an important prognostic parameter for the group of PCLBCL, leg type.

Cancer-associated mutations in chromatin remodeler hSNF5 promote chromosomal instability by compromising the mitotic checkpoint.

The hSNF5 subunit of human SWI/SNF ATP-dependent chromatin remodeling complexes is a tumor suppressor that is inactivated in malignant rhabdoid tumors (MRTs). Here, we report that loss of hSNF5 function in MRT-derived cells leads to polyploidization and chromosomal instability. Re-expression of hSNF5 restored the coupling between cell cycle progression and ploidy checkpoints. In contrast, cancer-associated hSNF5 mutants harboring specific single amino acid substitutions exacerbated poly- and aneuploidization, due to abrogated chromosome segregation. We found that hSNF5 activates the mitotic checkpoint through the p16INK4a-cyclinD/CDK4-pRb-E2F pathway. These results establish that poly- and aneuploidy of tumor cells can result from mutations in a chromatin remodeler.