Gene Expression Profiles in a Transgenic Animal Model of Fragile X Syndrome

Neurobiology of Disease 10, 211–218 (2002) doi:10.1006/nbdi.2002.0506

Gene Expression Profiles in a Transgenic Animal Model of Fragile X Syndrome Velia D’Agata,* ,† Stephen T. Warren, ‡ Weiqin Zhao,* Enrique R. Torre, ‡ Daniel L. Alkon,* and Sebastiano Cavallaro* ,† *Blanchette Rockefeller Neurosciences Institute, West Virginia University, Rockville, Maryland 20850; †Institute of Neurological Sciences, CNR, 95123 Catania, Italy; and ‡Howard Hughes Medical Institute and Departments of Biochemistry, Pediatrics, and Genetics, Emory University School of Medicine, Atlanta, Georgia 30322 Received November 9, 2001; revised March 27, 2002; accepted for publication April 12, 2002

Fragile X syndrome is the most common inherited form of mental retardation. Although this syndrome originates from the absence of the RNA-binding protein FMRP, the molecular mechanisms underlying the cognitive deficits are unknown. The expression pattern of 6789 genes was studied in the brains of wild-type and FMR1 knockout mice, a fragile X syndrome animal model that has been associated with cognitive deficits. Differential expression of more than two-fold was observed for the brain mRNA levels of 73 genes. Differential expression of nine of these genes was confirmed by real-time quantitative reverse transcription-polymerase chain reaction and by in situ hybridization. In addition to corroborating the microarray data, the in situ hybridization analysis showed distinct spatial distribution patterns of microtubule-associated protein 2 and amyloid beta precursor protein. A number of differentially expressed genes associated with the fragile X syndrome phenotype have been previously involved in other memory or cognitive disorders. © 2002 Elsevier Science (USA)

INTRODUCTION

sence of FMRP, could play a significant role in the cognitive deficits associated with fragile X syndrome. The generation of FMR1 knockout (KO) mice by homologous recombination (The Dutch-Belgian Fragile X Consortium, 1994) provides an animal model to test this hypothesis. Indeed, FMR1 knockout mice show macroorchidism, hyperactivity and behavioral deficits, abnormalities that mimic the human syndrome (The DutchBelgian Fragile X Consortium, 1994). To gain greater insight into the molecular mechanisms leading to mental retardation in fragile X syndrome, we have used the unprecedented experimental opportunities that the genome sequences and the development of cDNA array technology (Shena et al., 1995; Yue et al., 2001) now provide to perform genome-wide expression analysis.

Fragile X syndrome is the most common inherited form of mental retardation and originates from the loss of FMR1 expression due to trinucleotide repeat expansion (Fu et al., 1991; Verkerk et al., 1991). In addition to global cognitive deficits, the disorder can also be manifest as specific impairments in visualspatial learning as well as auditory and visual shortterm memory (Freund and Reiss, 1991; Fisch et al., 1996). Although the function of the FMR1 gene product, FMRP, is still unknown, the presence of three RNA binding regions (two KH domains and an RGB box) suggests that FMRP is an RNA-binding protein (Ashley et al., 1993; Siomi et al., 1993). Indeed, in vitro– translated FMRP has been demonstrated to preferentially bind certain RNA homopolymers and to selectively bind a subset of brain transcripts including its own message (Siomi et al., 1993). The observation that FMRP is an RNA binding protein and may be implicated in RNA metabolism suggests that other genes, whose products may vary in the ab0969-9961/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

EXPERIMENTAL PROCEDURES Animals. The subjects were six wild-type and six FMR1 knockout 4-month-old mice. Animals, housed

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212 two to six per cage, were given access to food and water, and maintained on a 12:12 light/dark cycle in a constant temperature (23°C). Animals were sacrificed by alothane overdose, decapitated and the brains rapidly removed and freezed in liquid nitrogen. The FMR1 knockout mice were generated by crossing a heterozygous FMR1 ko female with a FMR1 ko male, both derived from a cross between a wild-type C57B16/J male and a female heterozygous for the FMR1 ko allele (N11 congenic on C57B16/J). According to standard convention, N10 and beyond are considered fully congenic. Therefore the mice examined had little or no variation in strain background. Animals were genotyped by PCR using DNA obtained from tail clippings. The presence or absence of FMR1mRNA in the brain of wild-type and FMR1-knockout animals, respectively, was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) by the method previously described (The Dutch-Belgian Fragile X Consortium, 1994). cDNA microarray analysis. Poly(A)⫹ RNA was isolated from a pool of three brains from adult FMR1 knockout male mice and wild-type male littermates. The quality of RNA was verified by agarose gel electrophoresis and spectrophotometer readings (optical density 260/280 ⬎ 1.8). cDNA generation, hybridization to the Mouse GEM-1 cDNA microarray and data collection were performed by Incyte Genomics (Palo Alto, CA), as described at http://reagents.incyte.com/expression/ technology/index.html. In brief, alterations in gene expression were evaluated by reverse transcription of poly(A) RNAs in the presence of Cy3 or Cy5 fluorescent labeling dyes followed by hybridization to a mouse GEM-1 microarray chip containing a total of 125 control samples and 8732 mouse cDNAs. Of these, 3336 fall into annotated gene clusters (UniGene Mus Musculus database, Build 74). Microarray data were analyzed with GEMTools 2.5 (Incyte Genomics) and GeneSpring 4.2 (Silicon Genetics). Unigene (http://www.ncbi.nlm.nih. gov/UniGene), HomoloGene (http://www.ncbi.nlm. nih.gov/HomoloGene), LocusLink (http://www.ncbi. nlm.nih.gov/LocusLink/index.html), and the Mouse Genome Informatics at the Jackson Laboratory (http:// www.informatics.jax.org/) were used to obtain information on nomenclature, sequence accession, homology, and gene ontology. The following criteria were used to define genes with measurable levels of expression: (1) a minimum signal intensity of 100 in at least one of the two probes, (2) signal to background ratios greater than 2.3 for both probes, (3) signal size greater than 40% of the spotting area for both probes, (4) spotted DNA passed the PCR screening. A complete list of them is available 2002 Elsevier Science (USA) All rights reserved.

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FIG. 1. Microarray analysis of brains from wild-type and FMR1 knockout mice. Spots indicate the expression of 6789 genes showing measurable levels of expression. Red lines indicate the n fold (1, 2) change between signal values in wild-type versus FMR1 knockout mice.

online at http://www.brni-jhu.org/sebi/microarraydata/FMR1.htm as supplementary information. Real time quantitative RT-PCR. Following extraction, total RNA samples were reverse transcribed with oligo(dT) 12–18 and SuperScript II Rnase H-reverse transcriptase (GibcoBRL). Aliquots of cDNA (0.1 and 0.2 ␮g) and known amounts of external standard (purified PCR product, 10 2 to 10 8 copies) were amplified in parallel reactions using forward (FP) and reverse (RP) primers indicated in Table 2. To control for the integrity of RNA and for differences attributable to errors in experimental manipulation, mRNA levels of mouse ribosomal S18 and phosphoglycerate kinase 1 were measured in similar reactions. Each PCR reaction (final volume 20 ␮l) contained 0.5 ␮M of primers, 2.5 mM Mg 2⫹ and 1 X DNA SYBR Green master mix (Roche Molecular Biochemicals, Mannheim, Germany). To prevent nonspecific amplification “Hot Start” was performed by preincubating the DNA SYBR Green master mix with TaqStart Antibody solution (Clontech) for 5 min at RT. PCR amplifications were performed with a Light-Cycler (Roche Molecular Biochemicals, Mannheim, Germany) using the following four cycle programs: (i) denaturation of cDNA (1 cycle: 95°C for 1 min); (ii) amplification (40 cycles: 95°C for 0 s, 57°C for 5 s 72°C for 10 s); (iii) melting curve analysis (1 cycle: 95°C for 0 s, 67°C for 10 s, 95°C for 0 s); (iv) cooling (1 cycle: 40°C for 3 min). Temper-

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FIG. 2. In situ hybridization validation of microarray results. Specific riboprobes labeled with [␣- 35S] for MAP2- and APP-mRNAs were hybridized with brain sections of individual FMR1 knockout mice and wild type littermates. Labeled mRNA signals were revealed with autoradiography. The pictures in the left and right panel show the representative distribution of MAP2- and APP-mRNAs, respectively. The color spectrum on the right side of each panel represents the pixel value of gray levels. The bar graphs represent the mean ⫾ SD of optical density values in different brain areas from each group of four animals (*P ⬍ 0.01, **P ⬍ 0.003).

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TABLE 1 Differentially Expressed Genes Determined by Microarray Analysis in Brains of Wild-Type versus FMR1 Knockout Mice

Function

Signal transduction and regulation (878)

Metabolism (1426)

Structural proteins (1611)

Nucleic acid synthesis and modification (85)

Unknown

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Wild-type signal

FMR1 KO signal

Fold change

200 607 409 473 153 261 1124 464

73 270 195 225 72 541 2374 1146

⫺2.7 ⫺2.2 ⫺2.1 ⫺2.1 ⫺2.1 2.1 2.1 2.5

10206 172 355 3393 164 615

84 77 804 8954 473 10774

⫺121.5 ⫺2.2 2.3 2.6 2.9 17.5

7294 8749 348

88 94 154

⫺82.9 ⫺94.0 ⫺2.3

146 497 931 151 2740 2549 1845 541 5604 780 5279 600 783 514 200 22610 10885 10029 6102 368 398 1097 164 150 198 3281 381 283 171 186 140 156 137 158 145

67 241 436 75 5752 5301 3889 1137 12597 1752 11538 1354 1833 8851 3950 137 88 84 76 133 148 421 75 69 93 1578 182 137 83 87 66 74 67 78 72

⫺2.2 ⫺2.1 ⫺2.1 ⫺2.0 2.1 2.1 2.1 2.1 2.2 2.2 2.2 2.3 2.3 17.2 19.8 ⫺165.0 ⫺123.7 ⫺119.4 80.3 ⫺2.8 ⫺2.7 ⫺2.6 ⫺2.2 ⫺2.2 ⫺2.1 ⫺2.1 ⫺2.1 ⫺2.1 ⫺2.1 ⫺2.1 ⫺2.1 ⫺2.1 ⫺2.0 ⫺2.0 ⫺2.0

Gene name

Accession number

Ser/Thr kinase KKIAMRE Adrenergic receptor, beta 2 RAB, member of RAS oncogene family-like 3 Receptor activity modifying protein 2 SemaF cytoplasmic domain associated protein 2 Growth factor regulated calcium channel (GRC) DAP kinase 2 Pinch

AA414632.1 AI323188.1 AA177178.1 W98502.1 AA023463.1 AA015295.1 AI322362.1 AA289280.1

Ubiquitin specific protease 7 Glycogen synthase 2 Protein phosphatase 1, catalytic subunit, beta isoform Aminopeptidase P Carboxylesterase ESTs, Highly similar to ubiquitin-binding protein

AA444224.1 AA537291.1 AA285841.1 W83771.1 WI4805.1 AA388099.1

Microtubule-associated protein 2 (MAP2) ESTs, Highly similar to meningioma-expressed antigen 5 Imap38

AA386889.1 AA388172.1 AA275014.1

Ribonuclease P protein subunit p40 Werner Tripartite motif protein 10 Peptidylprolyl isomerase E (cyclophilin E) Nuclear receptor binding factor-1 (NRBF-I) RL/IF-1 Ets-1 Translation elongation factor 1 alpha 2 Myn Elongation factor 1-alpha (EF 1-alpha) Origin recognition complex, subunit 6-like INCENP Inner centromere protein Retinoic acid receptor alpha 2 ESTs, Highly similar to p105 coactivator EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST

AA444640.1 AA541835.1 AA032514.1 W89608.1 AA259674.1 AA250462.1 AA266478.1 AI327504.1 AA415602.1 AA259551.1 AA415516.1 AA034771.1 W62668.1 AA386847.1 AA413831.1 AA422809.1 AA414642.1 AA413752.1 AA414282.1 AA051252.1 AA276825.1 W36450.1 AA416091.1 AA389271.1 AA119353.1 AA050028.1 AA423601.1 AA146200.1 AA266097.1 W11146.1 AA288042.1 AA200358.1 AA198969.1 AA034697.1 AA212170.1

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Gene Expression Profiles of FMR1 Knockout Mice TABLE 1—Continued

Function

Unknown

Wild-type signal

FMR1 KO signal

Fold change

Gene name

Accession number

154 153 164 325 469 423 686 1119 1609 4658 6208 1146 5124 6768 250 2746 1028 134 157 614 930

76 76 81 162 233 852 1400 2264 3224 9616 12788 2526 11052 15896 624 7245 2780 1420 1698 12931 22782

⫺2.0 ⫺2.0 ⫺2.0 ⫺2.0 ⫺2.0 2.0 2.0 2.0 2.0 2.1 2.1 2.2 2.2 2.3 2.5 2.6 2.7 10.6 10.8 21.1 24.5

EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST EST

W13059.1 AA031238.1 AA260199.1 AA423205.1 AA545589.1 AA051666.1 AA266353.1 AA28584.1 AA275684.1 AA413678.1 AA465838.1 AA067119.1 AA422973.1 AA415519.1 W89614.1 AA087542.1 W59584.1 AA414028.1 AA415185.1 AA388125.1 AA387431.1

Note. The following criteria were used to define candidate differentially expressed genes: (1) a minimum signal intensity of 100 in at least one of the two probes; (2) signal to background ratios greater than 2.3 for both probes; (3) signal size greater than 40% of the spotting area for both probes; (4) spotted DNA passed the PCR screening; (5) fold change greater than 2. Known genes were grouped into functional classes. The total number of genes for each class is reported in parenthesis. The inclusion of ESTs in front of a gene name indicates that the gene is highly similar but not identical in sequence to the named gene. Observed hybridization signals for both probes is indicated.

ature transition rate was 20°C/s except for the third segment of the melting curve analysis where it was 0.2°C/s. Fluorimeter gain value was 7. Real-time detection of fluorimetric intensity of SYBR Green I, indicating the amount of PCR product formed, was measured at the end of each elongation phase. Quantification was performed by comparing the fluorescence of PCR products of unknown concentration with the fluorescence of the external standards. For this analysis, fluorescence values measured in the log-linear phase of amplification were considered using the second derivative maximum method of the Light Cycler Data Analysis software (Roche Molecular Biochemicals, Mannheim, Germany). Specificity of PCR products obtained was characterized by melting curve analysis followed by gel electrophoresis and DNA sequencing. In situ hybridization. Brains from FMR1 knockout male mice and wild type male littermates (n ⫽ 4) were rapidly frozen on dry ice, and brain sections (12 ␮M) were obtained with a cryostat set at ⫺20°C. In situ hybridization was performed according to procedures described previously (Beyreuther et al., 1992) for two mRNAs, microtubule-associated protein 2 (MAP2)

and amyloid beta precursor protein (APP). I.M.A.G.E. Consortium (LLNL) cDNA clones (Alves-Rodrigues et al., 1998) corresponding to MAP2 (GenBank Accession No. AA386889.1) and APP (GenBank Accession No. AA110872.1) were linearized with SalI and SpeI, respectively. Specific antisense riboprobes for MAP2 and APP were labeled with [␣- 35S] by using SP6 and T7 primer, respectively. No signal was detected in control brain sections hybridized with the sense riboprobe or pretreated with RNase before hybridization with the antisense probe. Signals were obtained with film autoradiography. Evaluation of hybridization signals were obtained by using a computer-assisted image analysis system and the NIH Image 1.49 software (Wayne Rasband, NIH, Bethesda, MD).

RESULTS AND DISCUSSION Messenger RNA levels from brains of control wildtype littermates and FMR1 knockout mice (n ⫽ 3 per group) were simultaneously analyzed with high-density cDNA microarrays containing 8731 mouse cDNA ©

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TABLE 2 Confirmatory Analysis of Differentially Expressed Genes by Quantitative RT-PCR

Gene name

Accession number

Ser/Thr kinase KKIAMRE

AA414632.1

SemaF cytoplasmic domain associated protein 2 Microtubule-associated protein 2 Werner

AA023463.1

ESTs, Unknown

AA414282.1

ESTs, Unknown

AA051252.1

ESTs, Unknown

AA388125.1

ESTs, Unknown

AA087542.1

Ribosomal protein S18

M31788

Phosphoglicerate Kinase 1

X57529

AA386889.1 AA541835.1

Copies/ng of RNA Primer sequences

Wild-type

FMR1 KO

Fold change

FP: 5⬘-GATCAATGAGGAGCATGGCT-3⬘ RP: 5⬘-AGTCATCATGTGTGCTCCACA-3⬘ FP: 5⬘-TCGCTCTTGACGAGACTCTG-3⬘ RP: 5⬘-GTCATCTCTGCAGGAACACG3⬘ FP: 5⬘-GAAGAGTTCCAAGGCCACTT-3⬘ RP: 5⬘-CAGGCAGATCCAGAGAGAGT-3⬘ FP: 5⬘-AGACCGAACCTCCTCCTCTG-3⬘ RP: 5⬘-CTCAATCTGCAAGATTCCATGT-3⬘ FP: 5⬘-AATGGTGACCGAGATCTGTG-3⬘ RP: 5⬘-CTCTCGCATAGGCACTTCTT-3⬘ FP: 5⬘-CCTGAGATGCCGACTACCTT-3⬘ RP: 5⬘-TAGTCTGGCCTGTTCACCTG-3⬘ FP: 5⬘-CCAGCACACCAAGGAGAGTA-3⬘ RP: 5⬘-CCTTCTCAGATGCTCACTGG-3⬘ FP: 5⬘-ACTGGACCTACTGACAGCCA-3⬘ RP: 5⬘-CTGAGCCACCTGAAGACAGT-3⬘ FP: 5⬘-CAGAAGGACGTGAAGGATGG-3⬘ RP: 5⬘-CAGTGGTCTTGGTGTGCTGA-3⬘ FP: 5⬘-AGGTGCTCAACAACATGGAG-3⬘ RP: 5⬘-TACCAGAGGCCACAGTAGCT-3⬘

488.1 ⫾ 42.7

216.7 ⫾ 41.8

⫺2.2*

447.5 ⫾ 51.4

189.2 ⫾ 22.2

⫺2.3*

1148.3 ⫾ 107

703.9 ⫾ 96.4

⫺1.6*

391.3 ⫾ 29

151.6 ⫾ 38.3

⫺2.5*

206.4 ⫾ 9.4

96.55 ⫾ 6.6

⫺2.1*

638.1 ⫾ 62

221.9 ⫾ 35.7

⫺2.8*

931.55 ⫾ 13.6

1692 ⫾ 43.1

1.8*

681.8 ⫾ 59.8

1252 ⫾ 32.7

1.8*

103140 ⫾ 23051

95965 ⫾ 17472

1

37415 ⫾ 3033

40060 ⫾ 2559

1

Note. Transcript levels for the indicated genes were assayed by real-time detection and evaluation of fluorimetric PCR reactions. Results are the mean ⫾ SD of absolute RNA levels (copy number) from wild-type and FMR1 knockout brains (data points were obtained from three individual animals, each run in triplicate, *P ⱕ 0.05).

clones with a length of 500 –5000 bp and with averages in the 1-kb region. Of the 6789 genes showing measurable levels of expression (Fig. 1), a change of more than twofold was observed for the mRNA levels of 73 genes (Table 1). Of these genes, 14 showed changes greater than 10-fold. Although our data represented the average gene expression in the brains of wild-type and FMR1 knockout mice, there could be differences in gene expression between individual animals. To address this question and confirm the microarray results, we selected 9 (12%) of the genes which were differently expressed and validated (P ⬍ 0.05) their differential expression by quantitative reverse transcription-polymerase chain reaction (RTPCR) (Table 2) and/or in situ hybridization (Fig. 2). Although cDNA microarray analysis and quantitative RT-PCR have different precision and sensitivity ranges, differential expression ratios generated by the two methods were concordant in showing the up- or down-regulation for all the genes tested. In three of eight cases (gene accession mumbers: AA386889.1, AA414282.1, and AA388125.1); however, the ratios generated by microarray analysis appeared overstated and could reflect the lower precision of this technology when high differential 2002 Elsevier Science (USA) All rights reserved.

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expression ratios are observed and one of the two signals is close to background levels (Yue et al., 2001). In addition to corroborating the microarray data, in situ hybridization analysis revealed distinct patterns of spatial distribution for the two genes tested, microtubule-associated protein 2 (MAP2) and amyloid beta precursor protein (APP). Figure 2 shows their regional mRNA expression in wild-type and FMR1 knockout mice. MAP2-mRNA was decreased in the cerebral cortex and hippocampus of FMR1 knockout mice. In the same animals, a marked increase of APP-mRNA was observed in the cerebral cortex, hippocampus and cerebellar cortex. Many of the differential expressed genes have no currently recognized function and are not yet named. Indeed, 41 of the differentially expressed genes have no apparent homology to any gene whose function is known. Complete nucleotide sequence determination, conceptual translation, expression monitoring, and biochemical analysis should provide a detailed functional understanding of these genes. The 32 remaining genes, however, do have significant similarity to known genes and can be grouped into four different classes (Table 1): (i) signal transduction and regula-

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tion, (ii) metabolism, (iii) structural proteins, and (iv) nucleic acid synthesis and modification. Although further tests are required to determine exactly what role, if any, these genes play in mental retardation, six of them have been previously related to memory or cognitive related disorders. Altered expression of MAP2 has been shown with contextual memory, long term potentiation, aging, epilepsy, Alzheimer’s disease, and Rett syndrome (Kosik et al., 1984; Johnson and Jope, 1992; Leterrier and Eyer, 1992; Kaufmann et al., 1995; Fukunaga et al., 1996; Yamanouchi et al., 1998; Woolf et al., 1999). MAP2 is heavily concentrated in mature dendrites and may be critical for dendritic stability (Johnson and Jope, 1992). Our microarray, quantitative PCR and in situ hybridization analysis indicate a down-regulation of MAP2-mRNA in FMR1 knockout animals. One previous study (Steward et al., 1998) reported no change in the relative distribution of MAP2-mRNA between the cell body and the dentritic laminae of the hippocampus. The study, carried out by nonisotopic in situ hybridisation, was undertaken to define the subcellular localization of MAP2-mRNA and did not provide a quantitative assessment of its levels. The Ser/Thr kinase KKIAMRE, whose expression is decreased in FMR1 knockout mice, is a cell division cycle 2-related protein kinase which has been shown to be induced following eyeblink conditioning (Gomi et al., 1999). Decreased expression of RAB, a member of the Rab small G protein family, was also observed in FMR1 knockout mice. Rab proteins are key regulators of vesicular transport, play critical roles in synaptic plasticity and their dysfunction has been linked to mental retardation phenotypes (Seabra et al., 2002). The Werner gene encodes a DNA helicase which is mutated in the Werner syndrome, an autosomal recessive genetic disorder that is manifested by accelerated aging as also reflected by extensive deposition of amyloid beta peptide in the central nervous system (Leverenz et al., 1998). In FMR1 knockout mice, we also observed increased mRNA expression of the APP, whose altered expression has been extensively linked to Alzheimer’s disease and Down syndrome (Jiang et al., 1999). Finally, altered expression of two proteins involved in the ubiquitin-proteosome protein degradation pathway, the ubiquitin-specific protease 7 and the ubiquitin-binding protein homolog, was found in the brains of FMR1 knockout mice. Abnormality in the ubiquitin system has been demonstrated in other cognitive disorders, such as Alzheimer’s disease and the Angelman syndrome (Zhao et al., 2000; Lennon et al., 1996).

It should be emphasized that the microarray provides estimates of changes in mRNA levels that cannot be correlated with the amount and function of the gene products. Translation and post-translational modifications of many gene products and protein turnover have dramatic effects on function, and these cannot be inferred from expression analysis alone. Nevertheless, the data reported here provide new information on the gene expression changes that that result from a deficiency of FMRP. The value of these experiments will progressively increase as more is learned about the function of each gene. Systematic characterization of expression patterns associated with the fragile X syndrome and other cognition related disorders, will provide a framework for interpreting the biological significance of the expression patterns observed in the present work. One of the main goals will be to use cDNA microarrays to classify these disorders based on shared gene expression patterns. Although characterized by unique phenotypic expression, cognitive disorders, nevertheless, may share common features as illustrated here by MAP2 and APP. The methods used here may also allow the identification of common genes associated with the mental retardation phenotype, and thereby provide targets for therapeutic intervention.

ACKNOWLEDGMENTS S.T.W. is an investigator of the Howard Hughes Medical Institute. This study was supported in part by Comitato Telethon Fondazione Onlus Grant E646 to S.C.

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