A small 12-kb haplotype upstream of the AKT1 gene has been found to be associated with insulin resistance phenotypes. We sought to define the functional consequences of the three component polymorphic loci (rs1130214, rs10141867, rs33925946) on AKT1 and the upstream ZBTB42 gene. 5′ RACE analysis of AKT1 transcripts in human skeletal muscle biopsies showed the predominant promoter to be 2.5 kb upstream of exon 2, and distinct from those promoters previously reported in rat. We then studied the effect of each of the three haplotype polymorphisms in transcriptional reporter assays in muscle, bone, and fat cell culture models, and found that each modulated enhancer and repressor activity are in a cell-specific and differentiation-specific manner. Our results in promoter assays are consistent with the human phenotype data; we found an anabolic effect on muscle and bone with increased mRNA expression of AKT1, and catabolic effect on fat with decreased expression. To test the hypothesis that rs10141867 affects transcription levels of the novel zinc finger protein ZBTB42 in vivo, we developed the allele-specific expression assay using Taqman technology to test for allelic differences within heterozygotes. The allele containing the derived polymorphism (haplotype H2) showed a 1.75-fold increase in expression in human skeletal muscle. Our data show a particularly complex effect of the component polymorphisms of a single haplotype on cells and tissues, suggesting that the coordination of different tissue-specific effects may have driven selection for the H2 haplotype. In light of the recent abundance of SNP association studies, our approach can serve as a method for exploring the biological function of polymorphisms that show significant genotype/phenotype associations.
AIM: To investigate the expression and prognostic value of carbonic anhydrase II (CA II) and Ki-67 in gastrointestinal stromal tumors (GISTs).
METHODS: One hundred and thirteen GIST patients admitted to Chinese People’s Liberation Army General Hospital from January 2004 to December 2010 were retrospectively followed up, and immunohistochemistry was used to detect CA II, Ki-67 and CD117 expression in tumor samples. The survival rates of the patients were analyzed using the Kaplan-Meier method. Log-rank test, χ2 test and Cox proportional hazards model were used to determine the relationships between CA II, Ki-67 and CD117 expression and prognostic value in GISTs.
RESULTS: The survival rates at 1, 3 and 5 years were 90.0%, 82.0% and 72.0% in all patients. However, in patients with positive CA II or Ki-67, the survival rates were 92.0%, 83.0% and 77.0% or 83.0%, 66.6% and 53.0%, respectively. Compared with the negative groups, the survival rates in the positive groups were significantly lower (CA II log-rank P = 0.000; Ki-67 log-rank P = 0.004). Multivariate Cox analysis revealed that CA II, CD117 and Ki-67 were considerable immune factors in prognosis of GIST patients (CA II P = 0.043; CD117 P = 0.042; Ki-67 P = 0.007). Besides, tumor diameter, mitotic rate, tumor site, depth of invasion, complete resection, intraoperative rupture, and adjuvant therapy were important prognosis predictive factors. Our study indicated that CA II had strong expression in GISTs and the prognosis of GISTs with high CA II expression was better than that of GISTs with low or no expression, suggesting that CA II is both a diagnostic and prognostic biomarker for GIST.
CONCLUSION: CA II and Ki-67 are significant prognostic factors for GISTs. CA II associated with neovascular endothelia could serve as a potential target for cancer therapy.
Gastrointestinal stromal tumors; Carbonic anhydrase; CD117; Ki-67; Prognostic factor
In the title molecule, C16H25NO4, the non-H atoms, except for the two tert-butyl groups, are roughly planar (r.m.s. deviation of the non-H atoms = 0.086 Å). In the crystal, molecules are linked into inversion dimers by pairs of N—H⋯O hydrogen bonds, forming R
2(10) ring motifs.
In the title molecule, C13H19NO4, except for two C atoms of the tert-butyl group, the non-H atoms are almost coplanar (r.m.s. deviation = 0.2542 Å). In the crystal, molecules are linked into centrosymmetric dimers by two intermolecular N—H⋯O hydrogen bonds, forming an R
2(10) ring motif.
AIM: To investigate the histological origin of pseudomyxoma peritonei (PMP) in Chinese women.
METHODS: The clinical and pathological data were reviewed for 35 women with PMP, and specimens of the peritoneal, appendiceal and ovarian lesions of each patient were examined using the PV-6000 immunohistochemistry method. Antibodies included cytokeratin (CK)7, CK20, mucin (MUC)-1, MUC-2, carbohydrate antigen (CA)-125, estrogen receptor (ER), and progesterone receptor (PR).
RESULTS: Abundant colloidal mucinous tumors were observed in the peritoneum in all 35 cases. Thirty-one patients had a history of appendectomy, 28 of whom had mucinous lesions. There was one patient with appendicitis, one whose appendix showed no apparent pathological changes, and one with unknown surgical pathology. Ovarian mucinous tumors were found in 24 patients. The tumors were bilateral in 13 patients, on the right-side in nine, and on the left side in two. Twenty patients had combined appendiceal and ovarian lesions; 16 of whom had undergone initial surgery for appendiceal lesions. Four patients had undergone initial surgery for ovarian lesions, and relapse occurred in these patients at 1, 11, 32 and 85 mo after initial surgery. Appendiceal mucinous tumors were found in each of these four patients. Thirty-three of the 35 patients showed peritoneal lesions that were positive for CK20 and MUC-2, but negative for CK7, MUC-1, CA125, ER and PR. The expression patterns in the appendix and the ovary were similar to those of the peritoneal lesions. In one of the remaining two cases, CK20, CK7 and MUC-2 were positive, and MUC-1, CA125, ER and PR were negative. The ovaries were not resected. The appendix of one patient was removed at another hospital, and no specimen was evaluated. In the other case, the appendix appeared to be normal during surgery, and was not resected. Peritoneal and ovarian lesions were negative for CK20, MUC-2, CK7, MUC-1, CA125, ER and PR.
CONCLUSION: Most PMP originated from the appendix. Among women with PMP, the ovarian tumors were implanted rather than primary. For patients with PMP, appendectomy should be performed routinely. The ovaries, especially the right ovaries should be explored.
Pseudomyxoma peritonei; Peritoneum; Tumor origin; Ovary; Appendix; Immunohistochemistry
In the title molecule, C8H11NO2, the r.m.s. deviation of non-H atoms from their best plane is 0.031 Å. Molecules are connected via a pair of N—H⋯O hydrogen bonds into a centrosymmetric dimer.
In the title compound, C16H18N4O3·2H2O, the dihedral angle between the pyrrole and pyridine rings in the hydrazone molecule is 7.12 (3)°. In the crystal structure, intermolecular N—H⋯O, O—H⋯N and O—H⋯O hydrogen bonds link the hydrazone and water molecules into double layers parallel to (101). The crystal packing exhibits weak π–π interactions between the pyrrole and pyridine rings of neighbouring hydrazone molecules [centroid–centroid distance = 3.777 (3) Å]. The crystal studied was a non-merohedral twin, the refined ratio of twin domains being 0.73 (3):0.27 (3).
Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.
GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.
We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.
Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.
The asymmetric unit of the title compound, C38H37N7O2, contains one half-molecule, situated on a twofold rotational axis, in which one amino group is involved in intramolecular N—H⋯O hydrogen bond and the two phenyl rings are twisted from the plane of pyrazolone ring by 26.69 (10) and 79.64 (8)°. The crystal packing exhibits no classical intermolecular contacts.
The relationships between the expression of CD133, Ki-67 and prognosis in gastric adenocarcinoma are unknown and needs exploring.
The samples of gastric adenocarcinoma from 336 Chinese patients with follow-up were analyzed for CD133 and Ki-67 protein expressions by immunohistochemical method.
CD133 was expressed in up to 57.4% (193/336) of this group of gastric carcinoma. The expression of CD133 was significantly higher in carcinoma than in normal (P = 0.0001) and dysplastic mucosas (P = 0.004). CD133 was positive corresponded with the tumour size, grade, infiltrative depth and clinical stage (all P < 0.05). The overall mean survival time of the patients with CD133 positive expression was shorter than that of patients with negative expression (P = 0.0001). The expression of CD133 has a positive correlation with that of Ki-67 (r = 0.188, P = 0.001) in gastric adenocarcinoma. CD133 was an independent prognostic indicator. (P = 0.0001).
It is suggested that CD133 may play an important role in the evolution of gastric adenocarcinoma and should be considered as a potential marker for the prognosis.
AIM: To explore the expression of leukemia related protein 16 (LRP16) in colorectal carcinoma, and analyze its correlation with clinicopathologic features and prognosis.
METHODS: Immunohistochemistry for LRP16 was performed in 201 cases of colorectal carcinoma and 60 cases of distal normal mucosa. Medical records were reviewed and clinicopathological analysis was performed.
RESULTS: LRP16 expression was detected in 117 of 201 cases of the colorectal carcinoma and in 21 cases of 60 distal normal mucosa. The expression of LRP16 in carcinoma was significantly higher than that in normal mucosa (χ2 = 9.999, P = 0.002). LRP16 protein expression was found in 43.3% (52/120) of carcinoma at stage I and II, and 80.2% (65/81) of carcinoma at stage III and IV (χ2 =27.088, P = 0.001). Correlation between LRP16 expression and clinicopathological factors was significant in differentiation (P = 0.010), tumor size (P = 0.001), infiltrative depth (P = 0.000) and distant metastasis (P = 0.027). The difference of median survival time between cancer patients with LRP16 expression (38.0 mo) and those without was statistically significant (105.0 mo, Log rank = 41.455, P = 0.001). The multivariate survival analysis revealed that LRP16 expression was correlated significantly (Cox’s regression: P = 0.001, relative risk = 2.082) with shortened survival in the patients with colorectal cancer.
CONCLUSION: The expression of LRP16 is related to the degree of differentiation, invasiveness, metastasis and prognosis of colorectal carcinoma.
Colorectal neoplasms; Immunohistochemistry; Leukemia related protein 16; Prognosis; Clinicopathology
AIM: To investigate the expression of leukemia related protein 16 (LRP16), and the possible relationship between LRP16 expression and clinicopathological indices in 336 gastric carcinoma patients.
METHODS: Immunohistochemistry was used to detect LRP16 expression in 336 cases of paraffin-embedded gastric carcinoma tissues and 60 cases of distal normal mucosa. The relationships between LRP16 expression and patients’ age, tumor size, histological grade, clinical stage, metastatic status and prognosis were analysed.
RESULTS: The expression of LRP16 was 58.6% (197/336) in gastric carcinoma and 31.7% (19/60) in distal normal gastric mucosa. The expression of LRP16 in carcinoma was significantly higher than that in normal mucosa tissues (χ2 = 14.929, P = 0.001). LRP16 protein expression was found in 44.1% (63/143) carcinomas at stage I and II, and 69.4% (134/193) carcinomas at stage III and IV (χ2 = 21.804, P = 0.001), and in 56.9% (182/320) of cancers without metastasis but 93.8% (15/16) of those with metastasis (χ2 = 8.543, P = 0.003). The expression of LRP16 was correlated with tumor size, infiltrative depth, clinical stage, lymphatic invasion and distant metastasis (all P < 0.05). Follow-up data showed that there was a significant difference in median survival time between cancer patients with expression of LRP16 (27.0 mo) and those without (48.0 mo, Log rank =31.644, P = 0.001).
CONCLUSION: The expression of LRP16 may be associated with invasion, metastasis and prognosis of gastric cancer.
Gastric neoplasms; Immunohistochemistry; Leukemia related protein 16; Prognosis
It is intuitive to speculate that nutrient availability may influence differentiation of mammalian cells. Nonetheless, a comprehensive complement of the molecular determinants involved in this process has not been elucidated yet. Here, we have investigated how nutrients (glucose) affect skeletal myogenesis. Glucose restriction (GR) impaired differentiation of skeletal myoblasts and was associated with activation of the AMP-activated protein kinase (AMPK). Activated AMPK was required to promote GR-induced transcription of the NAD+ biosynthetic enzyme Nampt. Indeed, GR augmented the Nampt activity, which consequently modified the intracellular [NAD+]/[NADH] ratio and nicotinamide levels, and mediated inhibition of skeletal myogenesis. Skeletal myoblasts derived from SIRT1+/− heterozygous mice were resistant to the effects of either GR or AMPK activation. These experiments reveal that AMPK, Nampt, and SIRT1 are the molecular components of a functional signaling pathway that allows skeletal muscle cells to sense and react to nutrient availability.
The relationships between the expression of ID2, EBV-LMP1 and P16(INK4A) in Chinese classical Hodgkin lymphoma are unknown and need exploring.
Samples of classical Hodgkin lymphoma from 60 Chinese patients were analyzed for the expression of ID2, EBV-LMP1 and p16(INK4A) proteins by immunohistochemistry.
ID2 protein was expressed in 83.3% of this group of classical Hodgkin lymphoma, staining strongly in both cytoplasm and nucleus of the Hodgkin and Reed-Sternberg (HRS) cells. EBV-LMP1 and P16(INK4A) were overexpressed in 85.0% and 71.7% of Hodgkin lymphoma, respectively. EBV-LMP1 was noted in the cytoplasm, membrane and nucleus of HRS cells; P16(INK4A) was in the nucleus and cytoplasm. Microscopically, ID2, EBV-LMP1 and P16(INK4A) staining distinguished the HRS cells from the complex background of lymphocytes. ID2 was positively correlated with EBV-LMP1(P < 0.01), but P16(INK4A) was inversely related to EBV-LMP1 (P < 0.05).
It is suggested that ID2, EBV-LMP1 and P16(INK4A) could play an important role in the evolution of classical Hodgkin lymphoma, and be considered as potential adjunct markers to identify HRS cells in diagnosis.
Network Component Analysis (NCA) has shown its effectiveness in discovering regulators and inferring transcription factor activities (TFAs) when both microarray data and ChIP-on-chip data are available. However, a NCA scheme is not applicable to many biological studies due to limited topology information available, such as lack of ChIP-on-chip data. We propose a new approach, motif-directed NCA (mNCA), to integrate motif information and gene expression data to infer regulatory networks.
We develop motif-directed NCA (mNCA) to incorporate motif information into NCA for regulatory network inference. While motif information is readily available from knowledge databases, it is a "noisy" source of network topology information consisting of many false positives. To overcome this problem, we develop a stability analysis procedure embedded in mNCA to resolve the inconsistency between motif information and gene expression data, and to enable the identification of stable TFAs. The mNCA approach has been applied to a time course microarray data set of muscle regeneration. The experimental results show that the inferred TFAs are not only numerically stable but also biologically relevant to muscle differentiation process. In particular, several inferred TFAs like those of MyoD, myogenin and YY1 are well supported by biological experiments.
A novel computational approach, mNCA, has been developed to integrate motif information and gene expression data for regulatory network reconstruction. Specifically, motif analysis is used to obtain initial network topology, and stability analysis is developed and applied with mNCA to extract stable TFAs. Experimental results on muscle regeneration microarray data have demonstrated that mNCA is a practical and reliable computational method for regulatory network inference and pathway discovery.
Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds, however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days, much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites, and tested candidate regulators in a 27 time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5′-CATTCCT-3′), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and, consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoD-Tead2-Fgfr4 pathway important for effective muscle regeneration.
Muscle regeneration; Tead; TEF; Fgfr; MyoD; Microarray
AIM: To observe the growth inhibitory effect of wild-type Kras2 gene on a colonic adenocarcinoma cell line Caco-2.
METHODS: Recombinant plasmid pCI-neo-Kras2 with wild type Kras2 open reading frame was constructed. The Caco-2 cells were transfected with either pCI-neo or pCI-neo-Kras2 using Lipofectamine 2000. The expression of wild type Kras2 was examined by Northern blot analysis. And the expression of wild type Kras2 protein was examined by Western blot analysis. The effects of wild-type Kras2 on cell proliferation were analyzed by monotetrazolium (MTT) assay, meanwhile analyses of cell cycle and spontaneous apoptosis rate were carried out by flow cytometry (FCM).
RESULTS: The plasmid of pCI-neo-Kras2 was successfully established. The growth rate of cells transfected with pCI-neo-Kras2 was significantly lower than the control cells transfected with the empty pCI-neo vector (P < 0.05). Cell cycle analysis revealed arrest of the pCI-neo-Kras2 transfected cells in G0/G1 phases, decreased DNA synthesis and decreased fractions of cells in S phase. The proliferative index of cells transfected with pCI-neo-Kras2 was decreased compared with the control cells (49.78% vs 64.21%)，while the apoptotic rate of Caco-2 cells with stable Kras2 expression increased (0.30% vs 0.02%).
CONCLUSION: The wild-type Kras2 gene effectively inhibits the growth of the colonic adenocarcinoma cell line Caco-2.
Colonic adenocarcinoma; Wild-type Kras2; Cell cycle; Apoptosis
AIM: To study the correlation between the patterns of subcellular expression of p16 and CDK4 in colorectal epithelia in the normal-adenoma-carcinoma sequence.
METHODS: Paraffin sections of 43 cases of normal colorectal epithelia and corresponding adenomas as well as carcinomas were analysed immunocytochemically for subcellular expression of p16 and CDK4 proteins.
RESULTS: Most carcinomas showed more cytoplasmic overexpression for p16 and CDK4 than the adenomas from which they arised or the adjacent normal mucosa. Most normal or non-neoplastic epithelia showed more p16 and CDK4 expression in the nucleus than their adjacent adenomas and carcinomas. There was a significant difference between the subcellular expression pattern of p16 and CDK4 in normal-adenoma-carcinoma sequence epithelia (P < 0.001). Neither p16 nor CDK4 subcellular patterns correlated with histological grade or Dukes' stage.
CONCLUSION: Interaction of expression of p16 and CDK4 plays an important role in the Rb/p16 pathway. Overexpression of p16 and CDK4 in the cytoplasm, as well as loss expression of p16 in the nucleus might be important in the evolution of colorectal carcinoma from adenoma and, of adenoma from normal epithelia.
Colorectal neoplasm; p16; CDK4; Immuno-cytochemistry
Autoimmune diseases are more prevalent in females than in males, whereas males have higher mortality associated with infectious diseases. To increase our understanding of this sexual dimorphism in the immune system, we sought to identify and characterize inherent differences in immune response programs in the spleens of male and female mice before, during and after puberty.
After the onset of puberty, female mice showed a higher expression of adaptive immune response genes, while males had a higher expression of innate immune genes. This result suggested a requirement for sex hormones. Using in vivo and in vitro assays in normal and mutant mouse strains, we found that reverse signaling through FasL was directly influenced by estrogen, with downstream consequences of increased CD8+ T cell-derived B cell help (via cytokines) and enhanced immunoglobulin production.
These results demonstrate that sexual dimorphism in innate and adaptive immune genes is dependent on puberty. This study also revealed that estrogen influences immunoglobulin levels in post-pubertal female mice via the Fas-FasL pathway.
AIM: To study the loss of heterozygosity (LOH) on 12p12-13 in Chinese colon carcinoma patients.
METHODS: DNA was extracted from 10 specimens of cancer tissue, 10 specimens of adjacent tissue and 10 specimens of normal tissue, respectively. LOH of Kras2 gene was analyzed by polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis using 11 microsatellite markers on 12p-12-13.
RESULTS: LOH of Kras gene was detected at least on one marker of 12p-12-13 in 30% (3/10) of adjacent tissue specimens. The highest frequency of LOH was identified on D12S1034 in 28.57% (2/7) of adjacent tissue specimens. LOH was detected at least on one marker of 12p12-13 in 60% (6/10) of carcinoma tissue specimens, the most frequent LOH was found on D12S1034 and D12S1591 in 42.86% (3/7) of carcinoma tissue specimens. LOH was detected in 30% (3/10) of carcinoma tissue specimens, 30% (3/10) of adjacent tissue specimens, and no signal in 1% (1/0) carcinoma tissue specimen. The occurrence of LOH did not correlate with sex, age, tumor size and lymph node metastasis.
CONCLUSION: Genomic instability may occur on 12p-12-13 of Kras2 gene in the development and progression of colon carcinoma. The high LOH of Kras2 gene may directly influence the transcription and translation of wild type Kras2 gene.
Colon carcinoma; Loss of heterozygosity; Kras2
Publicly accessible DNA databases (genome browsers) are rapidly accelerating post-genomic research (see http://www.genome.ucsc.edu/), with integrated genomic DNA, gene structure, EST/ splicing and cross-species ortholog data. DNA databases have relatively low dimensionality; the genome is a linear code that anchors all associated data. In contrast, RNA expression and protein databases need to be able to handle very high dimensional data, with time, tissue, cell type and genes, as interrelated variables. The high dimensionality of microarray expression profile data, and the lack of a standard experimental platform have complicated the development of web-accessible databases and analytical tools. We have designed and implemented a public resource of expression profile data containing 1024 human, mouse and rat Affymetrix GeneChip expression profiles, generated in the same laboratory, and subject to the same quality and procedural controls (Public Expression Profiling Resource; PEPR). Our Oracle-based PEPR data warehouse includes a novel time series query analysis tool (SGQT), enabling dynamic generation of graphs and spreadsheets showing the action of any transcript of interest over time. In this report, we demonstrate the utility of this tool using a 27 time point, in vivo muscle regeneration series. This data warehouse and associated analysis tools provides access to multidimensional microarray data through web-based interfaces, both for download of all types of raw data for independent analysis, and also for straightforward gene-based queries. Planned implementations of PEPR will include web-based remote entry of projects adhering to quality control and standard operating procedure (QC/SOP) criteria, and automated output of alternative probe set algorithms for each project (see http://microarray.cnmcresearch.org/pgadatatable.asp).
We provide a systematic study of the sources of variability in expression profiling data using 56 RNAs isolated from human muscle biopsies (34 Affymetrix MuscleChip arrays), and 36 murine cell culture and tissue RNAs (42 Affymetrix U74Av2 arrays).
We studied muscle biopsies from 28 human subjects as well as murine myogenic cell cultures, muscle, and spleens. Human MuscleChip arrays (4,601 probe sets) and murine U74Av2 Affymetrix microarrays were used for expression profiling. RNAs were profiled both singly, and as mixed groups. Variables studied included tissue heterogeneity, cRNA probe production, patient diagnosis, and GeneChip hybridizations. We found that the greatest source of variability was often different regions of the same patient muscle biopsy, reflecting variation in cell type content even in a relatively homogeneous tissue such as muscle. Inter-patient variation was also very high (SNP noise). Experimental variation (RNA, cDNA, cRNA, or GeneChip) was minor. Pre-profile mixing of patient cRNA samples effectively normalized both intra- and inter-patient sources of variation, while retaining a high degree of specificity of the individual profiles (86% of statistically significant differences detected by absolute analysis; and 85% by a 4-pairwise comparison survival method).
Using unsupervised cluster analysis and correlation coefficients of 92 RNA samples on 76 oligonucleotide microarrays, we found that experimental error was not a significant source of unwanted variability in expression profiling experiments. Major sources of variability were from use of small tissue biopsies, particularly in humans where there is substantial inter-patient variability (SNP noise).
We used expression profiling to define the pathophysiological cascades involved in the progression of two muscular dystrophies with known primary biochemical defects, dystrophin deficiency (Duchenne muscular dystrophy) and α-sarcoglycan deficiency (a dystrophin-associated protein). We employed a novel protocol for expression profiling in human tissues using mixed samples of multiple patients and iterative comparisons of duplicate datasets. We found evidence for both incomplete differentiation of patient muscle, and for dedifferentiation of myofibers to alternative lineages with advancing age. One developmentally regulated gene characterized in detail, α-cardiac actin, showed abnormal persistent expression after birth in 60% of Duchenne dystrophy myofibers. The majority of myofibers (∼80%) remained strongly positive for this protein throughout the course of the disease. Other developmentally regulated genes that showed widespread overexpression in these muscular dystrophies included embryonic myosin heavy chain, versican, acetylcholine receptor α-1, secreted protein, acidic and rich in cysteine/osteonectin, and thrombospondin 4. We hypothesize that the abnormal Ca2+ influx in dystrophin- and α-sarcoglycan–deficient myofibers leads to altered developmental programming of developing and regenerating myofibers. The finding of upregulation of HLA-DR and factor XIIIa led to the novel identification of activated dendritic cell infiltration in dystrophic muscle; these cells mediate immune responses and likely induce microenvironmental changes in muscle. We also document a general metabolic crisis in dystrophic muscle, with large scale downregulation of nuclear-encoded mitochondrial gene expression. Finally, our expression profiling results show that primary genetic defects can be identified by a reduction in the corresponding RNA.
muscular dystrophy; microarray; dystrophin; α-sarcoglycan; expression profiling