Human Molecular Genetics

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Human Molecular Genetics, 2003, Vol. 12, No. 6 595-600
© 2003 Oxford University Press

cDNA microarray analysis of individual Duchenne muscular dystrophy patients

Satoru Noguchi^1,2, Toshifumi Tsukahara^1,*, Masako Fujita^1,2, Rumi Kurokawa^1,2, Masaji Tachikawa^2,3, Tatsushi Toda^2,3, Atsumi Tsujimoto^2,4, Kiichi Arahata^1,2, and Ichizo Nishino^1,2

¹Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan, ²Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan, ³Division of Functional Genomics, Department of Post-Genomics and Diseases, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan and ⁴DNA Chip Research Inc, Yokohama, Kanagawa 230-0045, Japan

Received October 1, 2002; Accepted January 17, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
We have developed a novel cDNA microarray encompassing 3500 genes expressed in skeletal muscle. With this system, we have performed the first study of gene expression in samples from individual patients. We analyzed muscle specimen from individuals with Duchenne muscular dystrophy to identify differences among patients. Among the variably expressed genes, we focused on the expression of the genes encoding HLA-related proteins, myosin light chains and troponin Ts as markers of muscle necrosis and regeneration. The expression patterns of these genes correlated with the severity of dystrophic changes on histological examination. Our cDNA microarray provides a new tool to investigate molecular muscle pathology.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Dystrophin gene mutations cause dystrophin deficiency in sarcolemma giving rise to Duchenne muscular dystrophy (DMD) (1). Dystrophin is associated with several sarcolemmal proteins that connect the cytoskeleton and extracellular matrix (2). This connection bolsters the mechanical strength of the sarcolemma against muscle contraction-induced tension (3). In muscle lacking dystrophin, the connection is weakened and sarcolemma is exposed to high tension. It causes partial disruptions of sarcolemma leading to an influx of extracellular Ca²⁺. The elevation of intracellular Ca²⁺ concentration activates the calcium-dependent degradative pathway resulting in myofibril disruption and muscle necrosis (4). However, this mechanism has not been proven definitively.

It has been postulated that dystrophin is involved in signal transduction, because dystrophin has multiple phosphorylation sites and the dystrophin-associated proteins bind to Grb2, ERK6 and nNOS (5–7). Moreover, DMD muscles also show hypertrophy in some myofibers. To account for this muscle hypertrophy, two possible mechanisms have been proposed: abnormal IGF-1 and GDF8-mediated signal pathways (8,9). To clarify the mechanisms causing dystrophic changes and muscle hypertrophy in DMD, it is necessary to understand the molecular events that occur in dystrophic muscle. cDNA microarray/gene chip analyses provide comprehensive quantitative assays for transcripts and have been broadly applied to assess alterations of gene expression in diseases (10).

DMD and -sarcoglycanopathy have been analyzed by cDNA microarray/gene chip technique. The data clearly reveal expression profiles that are characteristic of DMD and -sarcoglycanopathy (11,12). However, these analyses have several limitations: (1) the cDNA probes plotted on the microarrays do not cover all of the genes expressed in skeletal muscle; (2) the properties of probe cDNAs have not been not well-characterized; (3) homologous genes of each target gene may cross-hybridize with the probes; and (4) because relatively large amounts of RNA are required, each microarray analysis has required pooled RNA samples from several patients (11). To resolve these problems, we developed a novel high-quality cDNA microarray and devised an analytical method. We applied our array to study gene expression in individual DMD patients in order to identify specific transcriptional changes related to the pathophysiological alterations in each patient.

	RESULTS

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Generation of a cDNA array collecting skeletal muscle transcripts
To generate novel cDNA microarray, we initially collected information about all genes related to skeletal muscle available through public databases. Based upon these data, we designed cDNAs probes. Blast searches were performed with each candidate probe to exclude the possibility of cross-hybridization with homologous genes. We identified cDNA fragments of 450–550 bp in size to minimize cross-hybridization. We made a cDNA microarray contained more than 4224 cDNA probes representing 3500 genes. This array showed significant reproducibility with a high correlation factor (>0.984), when target cDNAs, prepared from 1 µg of total RNA, were repeatedly hybridized. The hybridization spot intensities showed linearities of targets over a range that exceeded three-logs with 0.5–4 µg of total RNA (not shown).

cDNA microarray analysis with RNA from individual biopsy specimen
Histopathological features were assessed in each patient, because the severity of dystrophic phenotype differed among the patients. Because of the pathological variability, we thought that the expression analyses with the cDNA microarray should be performed on individual patients. Therefore, we developed methods to isolate and quantitate total RNA from small biopsy specimens and employed an enhanced detection system using tyramide signal amplification. Our microarray system first enabled us to analyze a gene expression profile in only 10–30 mg of frozen muscle and thus study a sample from a single patient. To confirm the reliability of our cDNA microarray, we analyzed the expression levels of whole transcripts in normal skeletal muscle. The cDNA probes were ordered according to their mean hybridized intensities in duplicate analyses. Probes derived from the same genes showed similar intensities. The ranking of genes matched well with those of EST abundance reported by Bortoluzzi et al. (13) and BODYMAP (14), thus demonstrating compatibility of our data with those with other methods. The ranking of genes expressed in skeletal muscle and its comparison are presented in supplemental data (Supplementary Table 1).

View this table: [in this window] [in a new window]   Table 1. Clinicopathological features of the patients

 
The common features of gene expression in DMD muscles
In this study, cDNA microarray analyses were carried out by competitive hybridization using probes from individual DMD and normal muscles, and the ratio of the both probe (signal intensitiy in DMD/signal intensitiy in control) in each experiment were estimated as differences in gene expression levels. We collected the data from the muscles of six patients and the complete sets of data are presented in Supplementary Table 2. To characterize the common features of gene expression in DMD muscles, average ratios for each spot in the six patients were calculated. Of the 3500 genes assessed by 4224 probes, 77 genes showed more than 2-fold increased expression in DMD muscle compared to controls, while 343 genes revealed decreased expression (less than 50% of controls) (Fig. 1). Table 1 shows the distribution of these upregulated and downregulated genes by functional classification. The complete sets of the variable genes are listed in supplementary data (Supplementary Tables 3 and 4). Genes related to immune response, sarcomere, extracellular matrix (ECM) and signaling/cell growth were increased predominantly. Most of the increased immune response-related genes are HLA-DR genes of major histocompatibility complex classes I and II, which are expressed in antigen-presenting cells. Upregulated sarcomere-related genes are -cardiac actin, myosin light chain (MYL) 4 and 1, and myosin binding protein H genes, which are known to be expressed developmentally in fetal muscles (15). Increased ECM genes are type III collagen -1, type XV collagen -1, SPARC and thrombospondin-4 genes. Upregulation of these genes reflect dystrophic changes in DMD muscles such as myofiber necrosis, inflammation and muscle regeneration. In contrast, genes involved in energy metabolism, transcription/translation, signaling and proteasomes were downregulated (Table 2). The genes in energy metabolism encode several enzymes involved in glycolytic pathway and electron transfer reactions in mitochondria (Supplementary Table 4). The genes in transcription/translation category encode transcriptional factors, initiation/elongation factors and ribosomal proteins, which are closely related to protein synthesis. Thus, downregulation of these genes may reflect the chronic decline in muscle function and homeostasis. Furthermore, our study revealed additional interesting findings. Genes encoding sarcomeric proteins were involved in both upregulated and downregulated groups. Interestingly, genes encoding skeletal and cardiac muscle actins and MYL 1, 3 and 4 isoforms, which are principal components in myofibrils, were strikingly upregulated. In contrast, genes encoding myofibril-associated or regulatory proteins, myosin-binding protein C, myomesin, titin, nebulin, desmin, telethonin and calpain 3 were downregulated. The genes expressed in slow fibers also tended to be downregulated.

View this table: [in this window] [in a new window]   Table 2. Classification of the genes representing variable expression more than 2-fold or less than 1/2-fold

 

View larger version (36K): [in this window] [in a new window]   Figure 1. Scatter plot of the average expression intensities on all spots between DMD patients and control. The diagonal line shows similar expression between DMD patients and control. The lines showing the 2-fold and 1/2-fold differences also indicated; 90.5% of probes present between the two lines.

 
Differences in gene expression in muscles among the patients
DMD is a progressive disorder and muscles from patients show a rather wide range of pathological changes depending on the stage of the illness. We monitored these pathological changes by histological examination of muscle sections from the patients (Fig. 2). The muscles from patients 4, 5 and 6 showed marked muscle fiber necrosis (asterisks) and regeneration (arrowheads) in addition to variations in fiber size and endomysial fibrosis (see also Table 1). In contrast, samples from patients 1, 2 and 3 showed much milder changes and these changes are least prominent in patients 2 and 3. We studied the difference in gene expression among the patients. Differences in gene expression in muscles among the patients should reflect the difference in the pathophysiological changes. Figure 3 shows the expression pattern of all probes in six patients in microarray analyses by Gene spring software from the data presented in Supplementary Table 2. Each of the horizontal color lines denotes the expression difference in each probe showing the upregulation in red or the downregulation in blue. All patients generally showed similar gene expression patterns, but in some probes the differences among the patients were remarkable. This experimental tree classified the patients into two groups, one consisting of patients 4, 5, and 6, and another containing patients 1, 2, and 3. This classification is essentially in accordance with the observation in histological examination in Figure 2 and Table 1.

View larger version (123K): [in this window] [in a new window]   Figure 2. Histological staining of skeletal muscle from DMD patients. Muscle sections show different degrees of dystrophic changes with variable muscle necrosis (asterisks) and regeneration (arrowheads) by hematoxylin and eosin staining. Bar indicates 100 µm. (A) patient 1, (B) patient 2, (C) patient 3, (D) patient 4, (E) patient 5 and (F) patient 6.

 

View larger version (36K): [in this window] [in a new window]   Figure 3. cDNA microarray analyses of skeletal muscle of DMD patients. Differences in gene expression are shown in color as bottom scale (5-fold expression in red to 1/5-fold expression in blue). The data for each patient are shown in each vertical column with an experimental tree. The probes were classified and aligned based on the expression differences in all patients as shown in left tree. P1, patient 1; P2, patient 2; P3, patient 3; P4, patient 4; P5, patient 5; P6, patient 6.

 
We hypothesized that the changes in expression of some genes reflect the state of pathological process in the patients' muscles. Therefore, we focused on genes related to the muscle necrosis and regeneration. As markers for muscle necrosis, we selected HLA-related genes, TIMP3 and C-X-C chemokine Scyb14 genes, which are known to be expressed in the infiltrating cells, dendritic cells or lymphocytes during immune response (16). We found that the expression level of these genes differed among the patients as shown in Figure 4A. These genes were strongly upregulated in patients 1, 4, 5 and 6, as indicated by red colors, while they were normally expressed in patients 2 and 3. These results indicate that patients 1, 4, 5, and 6 have more severe necrotic changes than patients 2 and 3, and are in accordance with the histological results shown in Figure 2. This observation was also confirmed by the differential expression of basic FGF, another marker for muscle degeneration among these patients (data not shown) (17).

View larger version (42K): [in this window] [in a new window]   Figure 4. Differential expression of HLA-related, MYL and TNNT genes in skeletal muscle of DMD patients. (A) The expression differences in probes with HLA-related genes. (B) Probes with MYL family genes. (C) Probes with TNNT family genes. The data are represented as in Figure 3. P1, patient 1; P2, patient 2; P3, patient 3; P4, patient 4; P5, patient 5; P6, patient 6.

 
We further hypothesized that regenerating process in muscle fibers will reproduce the course of expression of developmentally activated genes. We observed that several genes encoding muscle proteins, such as myosin heavy chains, MYLs, tropomyosin, myosin binding protein H, troponins, muscle creatine kinase, and myoglobin, showed the differential expression among patients (15). Among these genes, we found two gene families, MYL and troponin T (TNNT) which showed clear differences in expression between patients but also showed the distinct expression patterns between their gene isoforms. Figure 4B shows the expression levels of MYL gene family among six patients. In patients 5 and 6 with intensive muscle regeneration on histological examination, all MYL isoform genes were strongly expressed as shown in red signals. In patients 1, 2 and 3, MYL4, MYL1 and MYL3 genes were mildly upregulated as demonstrated in yellow to red signals; however, MYL2 was expressed much less in patients than in controls as indicated by in blue signals. Similarly, Figure 4C shows the expression levels of TNNT gene family among six patients. Patients 5 and 6 showed significant upregulation of all TNNT genes, and patient 4 showed slight expression of TNNT2 and TNNT3, while patients 1, 2 and 3 showed only faint expression in TNNT2 as shown in gray. We also confirmed the differential expression of marker proteins for regeneration, NCAM and myogenin (18) among these patients (data not shown). Overall, our results suggest that transcripts from three groups of genes, HLA-related, MYL and TNNT are useful reporters for monitoring dystrophic changes at the molecular level.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
With our novel microarray, we performed the first gene expression studies in muscle samples from individual patients. The analysis of DMD muscles from individual patients provided information about gene expression profile not only to deduce common pathognomonic features but also to highlight differences among patients at the molecular level. In common pathognomonic features in gene expression alteration, we found that developmentally expressed genes were significantly upregulated while the genes related to muscle homeostasis were downregulated. These changes reflect various cellular events including muscle necrosis and regeneration. These up- and downregulated genes are compatible with the results by Chen et al. (11) and Tkatchenko et al. (12). In addition, genes encoding principal components in myofibrils were strikingly upregulated while genes encoding myofibril-associated or regulatory proteins were downregulated, suggesting that these gene groups might be regulated by different mechanism, although only limited information is available about the promoters of some genes in these groups. We found that the genes expressed in slow-type fiber were also downregulated. The downregulation of these genes accompanied with the downregulation of mitochondrial energy metabolic genes in DMD muscles. This finding is interesting on the relation with recent report in which the activation of transcriptional complexes containing of PGC-1 in contractile stimulation upregulates the slow-type fiber genes and mitochondrial energy metabolic genes (19).

We have demonstrated the expression of genes related to muscle necrosis and regeneration among the patients. We detected the upregulation of HLA-related genes, TIMP3 and C-X-C chemokine Scyb14 genes dependent on the severity of necrotic changes in each patient's muscle. Their differential expression would indicate the degree of infiltration, activation of monocytes and lymphocytes and inflammatory response in necrotic areas of muscle among the patients (16). As muscle regeneration markers, we found the differential upregulation of MYL and TNNT isoform genes among patients dependent on the degrees of muscle regeneration in DMD muscle fibers. Progressive expression of each isoform in these families during mouse skeletal muscle development has also been reported (20). In the MYL gene family, MYL4 gene is activated initially, followed by sequential activation of the MYL1 and MYL3 genes during embryonic stages, while MYL2 is only expressed after birth (21). In the TNNT gene family, TNNT2 is expressed initially, and later TNNT3 and TNNT1 are expressed during mouse skeletal muscle development (22). The differential upregulation of MYL and TNNT gene isofoms among patients were well-matched with the progression of the muscle regenerating process, and reproduced the developmental expression patterns of these genes. Our data suggest that slow type genes, MYL2 and TNNT1 are the most sensitive marker of muscle fiber regeneration.

Our microarray data are in accordance with the histological abnormalities suggesting that this system may be used to monitor pathological changes in dystrophic muscles. Since our microarray requires only a small amount of muscle tissue, this system might contribute to providing additional information to histological analyses for the diagnosis and the evaluation of the muscular diseases, especially those with patchy necrotic and regenerating lesions, such as polymyositis in which multiple punch biopsies would help to make a diagnosis. Comprehensive gene expression analysis will also provide valuable information about mechanisms of muscle degeneration, degrees of progression or interruption of muscle regeneration, and differences at the molecular level among patients and among diseases. Our cDNA microarray will open a door to a new molecular pathology in the field of myology.

	MATERIALS AND METHODS

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Construction of the cDNA microarray
By database searches, we selected genes computationally including ESTs expressed in skeletal muscle and constructed probes for each predicted gene using the overlapping sequence. For each singleton, we designed one or more probes with unique sequence with lengths of 450–550 bp in the vicinity of 3'-ends of each transcript in order to obtain almost same hybridized condition for all spots and to minimize the possible cross-hybridization to other genes. We amplified cDNA fragments using specific primer pairs and cloned the PCR products into a pCR/blunt vector (Invitrogen, Carlsbad, CA, USA). All cloned products were confirmed by cycle sequencing on ABI prism 3100 (Applied Biosystems, Foster City, CA, USA). The inserts were amplified as probes with vector-arm primers, normalized in amounts and spotted on slideglass pre-coated with poly-L-lysine.

Preparation of total RNA from skeletal muscle biopsy specimens
Informed consent was obtained from all subjects. All patients used in this study were clinicopathologically diagnosed as DMD and all of their muscles showed no dystrophin-staining. The clinicopathological features of the patients are listed in Table 1. Muscle biopsy was carried on from biceps brachii muscles at ages as follows: patient 1, 3 years; patient 2, 1 year; patient 3, 1 year 6 months; patient 4, 1 year 10 months; patient 5, 5 years; patient 6, 4 years. We prepared total RNA from the 50–100 cryosections (6 µm thickness) of each muscle biopsy specimen with Cyto-plasmic RNA reagent (Invitrogen) according to the manufacturer's protocol. The amounts of total RNA were estimated by semi-quantitative RT–PCR with an intron-spanning primer pair for muscle creatine kinase coding region using the following used primer sets 5'-TTCATGTGGAACCAACACC-3' (811–829) and 5'-CAGAATCCAGAGGATGGAGC-3' (1334–1315). The amplified products from stepwise-diluted sample were compared with those from stepwise-diluted standard RNA from normal muscle.

Histological examination of skeletal muscle specimens
Hematoxylin and eosin staining and immunohistochemical staining of cryosections of biopsy specimens from patient skeletal muscles were performed by standard method.

Microarray analyses
The production of labeled cDNA targets, hybridization and detection on microarray were performed with TSA Labeling and Detection Kit (Perkin-Elmer Life Science, Boston, MA, USA) according to the manufacturer's protocol. We used approximately 1–2 µg of total RNA for labeling and used aliquots of the labeled products corresponding to 1 µg of total RNA for hybridization. The microarray experiments were carried out by competitive hybridization using two labeled targets from DMD muscle and normal one. Patients 3, 4, 5 and 6 were analyzed in duplicate experiments and patients 1 and 2 were analyzed only in single experiments because of the limited amounts of specimens. The hybridized intensities on spots and the background intensities around spots were quantitated by ScanArray 5000 equipped with the software of ScanArray and QuantArray (Perkin-Elmer Life Science). Difference in gene expression was represented as ratio of the fluorescent intensity with DMD target to that of normal control target on each spot. The data preparation and statistical analysis were carried out with Microsoft Excel (Microsoft, Redmond, WA, USA) and Genespring (Silicon Genetics, Redwood City, CA, USA). The mean intensity (x) and standard deviation () of the non-spotted areas without probes were calculated for each array, and the data of spots with the intensity value more than x+5 with at least one of two probes were used for statistical analyses. To deduce common features of gene expression in DMD muscles, we calculated the average ratio of each probe in all six patients' data and ranked the all genes according to these ratios using a median value if muliple probes from one gene were present. To compare expression profiling among DMD patients, we classified all probes using the ratio values in all patients' data with standard correlation at separation ratio 0.5 and minimum distance 0.001 by Gene tree classification and also classified the patients using all data by experimental tree classification in Genespring software.

	SUPPLEMENTARY MATERIAL

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Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
The authors thank Drs Y.K. Hayashi and I. Nonaka (National Center of Neurology and Psychiatry) for their critical comments and discussion on the manuscript, Dr M. Hirano (Columbia University) for reviewing the manuscript, Ms A. Nishiyama and S. Matsuno for their technical assistance.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +81 423412712; Fax: +81 423461742; Email: tukahara{at}ncnp.go.jp

Deceased.

	REFERENCES

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