Transcriptional abnormality in myotonic dystrophy affects DMPK but not neighboring genes

Myotonic dystrophy (DM) is caused by the expansion of a trinucleotide repeat, CTG, in the 3′ untranslated region of a protein kinase gene, DMPK. We set out to determine what effect this expanded repeat has on RNA processing. The subcellular fractionation of RNA and the separate analysis of DMPK transcripts from each allele reveals that transcripts from expanded DMPK alleles are retained within the nucleus and are absent from the cytoplasm of DM cell lines. The nuclear retention of DMPK transcripts occurs above a critical threshold between 80 and 400 CTGs. Further analysis of the nuclear RNA reveals an apparent reduction in the proportion of expansion-derived DMPK transcripts after poly(A)+ selection. Quantitative analysis of RNA also indicates that although the level of cytoplasmic DMPK transcript is altered in DM patients, the levels of transcripts from 59 and DMAHP, two genes that immediately flank DMPK, are unaffected in DM cell lines.

A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

Interleukin 12 deficiency associated with recurrent infections

A 3-yr-old female patient exhibited interleukin 12 (IL-12) deficiency that was associated with recurrent episodes of pneumococcal pneumonia with sepsis and other infections in the absence of fevers. The patient’s peripheral blood mononuclear cells (PBMCs) exhibited normal proliferative responses to antigens. Immune responses, including in vivo production of antibodies to diphtheria, tetanus, or pneumococcal antigens, were normal. Ig levels and B cell and T cell phenotypes were also normal. In contrast, IL-12 p70 heterodimer production was undetectable by using supernatants of the patient’s stimulated PBMCs when compared with control cells treated similarly. Although present, interferon γ (IFN-γ) was reduced. The addition of recombinant IFN-γ to control cells enhanced the production of IL-12 by up to sixfold. By contrast, IL-12 was undetectable in supernatants of the patient’s cells in the presence of recombinant IFN-γ. IL-12 p40 subunit mRNA by using the patient’s PBMCs after stimulation with Staphylococcus aureus Cowan strain 1 or lipopolysaccharide was also undetectable by reverse transcription–PCR when compared with control cells. Production of IL-2, IL-6, tumor necrosis factor α, or IFN-γ of the patient’s PBMCs after appropriate stimulation was observed. This patient has either a defect in Staphylococcus aureus Cowan strain 1-lipopolysaccharide- or staphylococcal enterotoxin A-induced signaling pathways for the activation of IL-12 p40 gene expression, or an abnormality in the IL-12 p40 gene itself.

Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 ζ chain of T-cell receptor complex and antigen-specific T-cell responses

One of the important mechanisms of immunosuppression in the tumor-bearing status has been attributed to the down-modulation of the CD3 ζ chain and its associated signaling molecules in T cells. Thus, the mechanism of the disappearance of CD3ζ was investigated in tumor-bearing mice (TBM). The decrease of CD3ζ was observed both in the cell lysate and intact cells. Direct interaction of T cells with macrophages from TBM (TBM-macrophages) induced the decrease of CD3ζ, and depletion of macrophages rapidly restored the CD3ζ expression. We found that treatment of such macrophages with N-acetylcysteine, known as antioxidant compound, prevented the decrease of CD3ζ. Consistent with this result, the addition of oxidative reagents such as hydrogen peroxide and diamide induced the decrease of CD3ζ expression in T cells. Consequently, the loss of CD3ζ resulted in suppression of the antigen-specific T-cell response. These results demonstrate that oxidative stress by macrophages in tumor-bearing status induces abnormality of the T-cell receptor complex by cell interactions with T cells. Therefore, our findings suggest that oxidative stress contributes to the regulation of the expression and function of the T-cell receptor complex.

Brain activity in visual cortex predicts individual differences in reading performance

The relationship between brain activity and reading performance was examined to test the hypothesis that dyslexia involves a deficit in a specific visual pathway known as the magnocellular (M) pathway. Functional magnetic resonance imaging was used to measure brain activity in dyslexic and control subjects in conditions designed to preferentially stimulate the M pathway. Dyslexics showed reduced activity compared with controls both in the primary visual cortex and in a secondary cortical visual area (MT+) that is believed to receive a strong M pathway input. Most importantly, significant correlations were found between individual differences in reading rate and brain activity. These results support the hypothesis for an M pathway abnormality in dyslexia and imply a strong relationship between the integrity of the M pathway and reading ability.

Perturbation of the T cell repertoire in rheumatoid arthritis

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) β-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR β-chain sequences of 1 in 2.4 × 107 CD4 T cells. In RA patients, the median TCR β-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool—as in speciation—aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinoderm embryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

Fusion of the transcription factor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)-positive papillary renal cell carcinomas

The (X;1)(p11;q21) translocation is a recurrent chromosomal abnormality in a subset of human papillary renal cell carcinomas, and is sometimes the sole cytogenetic abnormality present. Via positional cloning, we were able to identify the genes involved. The translocation results in a fusion of the transcription factor TFE3 gene on the X chromosome to a novel gene, designated PRCC, on chromosome 1. Through this fusion, reciprocal translocation products are formed, which are both expressed in papillary renal cell carcinomas. PRCC is ubiquitously expressed in normal adult and fetal tissues and encodes a putative protein of 491 aa with a relatively high content of prolines. No relevant homologies with known sequences at either the DNA or the protein level were found.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanism-based pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA.