Gene Expression Profiling Identifies WNT7A As a Possible Candidate Gene for Decreased Cancer Risk in Fragile X Syndrome Patients

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Archives of Medical Research - (2010) -

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ORIGINAL ARTICLE

Gene Expression Profiling Identifies WNT7A As a Possible CandidateGene for Decreased Cancer Risk in Fragile X Syndrome Patients

Monica Alejandra Rosales-Reynoso,a Alejandra Berenice Ochoa-Hernandez,b

Adriana Aguilar-Lemarroy,c Luis Felipe Jave-Suarez,c Rogelio Troyo-Sanroman,d

and Patricio Barros-Nunezb

aDivision de Medicina Molecular, bDivision de Genetica, cDivision de Inmunologıa, CIBO, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco,

Mexico, dDepartamento de Fisiologıa, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico

Received for publication September 19, 2009; accepted January 25, 2010 (ARCMED-D-09-00440).

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Address reprint r

Genetica, AP 1-3838

IMSS, Sierra Mojada

Jalisco, Mexico; Ph

---; E-mail: pbar

0188-4409/10 $eseedoi: 10.1016/j.arcm

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Background and Aims. Although sporadic cases of cancer in patients with fragile Xsyndrome (FXS) have been reported, extensive studies carried out in Denmark and Finlandconcluded that cancer incidence in these patients is lower than in the general population. Onthe other hand, the FMR1 protein, which is involved in the translation process, is absent inFXS patients. Hence, it is reasonable to assume that these patients exhibit an abnormalexpression of some proteins involved in regulating tumor suppressor genes and/or onco-genes, thus explaining its decreased cancer frequency. We undertook this study to analyzethe expression of oncogenes and tumor suppressor genes in fragile X syndrome patients.

Methods. Molecular analysis of the FMR1 gene was achieved in 10 male patients andcontrols. Total RNA from peripheral blood was used to evaluate expression of oncogenesand tumor suppressor genes included in a 10,000 gene microarray library. Quantitativereal-time PCR was utilized to confirm genes with differential expression.

Results. Among 27 genes showing increased expression in FXS patients, only eight genesexhibited upregulation in at least 50% of them. Among these, ARMCX2 and PPP2R5Cgenes are tumor suppressor related. Likewise, 23/65 genes showed decreased expressionin O50% of patients. Among them, WNT7A gene is a ligand of the b-catenin pathway,which is widely related to oncogenic processes. Decreased expression of WNT7A wasconfirmed by quantitative RT-PCR. Expression of c-Myc, c-Jun, cyclin-D and PPARdgenes, as target of the b-catenin pathway, was moderately reduced in FXS patients.

Conclusions. Results suggest that this diminished expression of the WNT7A gene maybe related to a supposed protection of FXS patients to develop cancer. � 2010 IMSS.Published by Elsevier Inc.

Key Words: Fragile X syndrome, Tumor suppressor genes, Oncogenes, WNT7A, Cancer risk.

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NCIntroduction

Fragile X syndrome (FXS) is the most common inherited formof mental retardation and the second leading cause of mentalretardation after Down syndrome (1e2). The estimated prev-alence of mental retardation in Western countries is 2e3%; of

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equests to: Dr. Patricio Barros-Nunez, Division de

, Centro de Investigacion Biomedica de Occidente,

800, Col. Independencia, CP 44340, Guadalajara,

one: (þ52) (33) 3668-3000 ext. 31930; FAX:

[email protected]

front matter. Copyright � 2010 IMSS. Published by Elseved.2010.03.001

these, 25e35% may have a genetic background. Among thegenetic causes, almost one third are probably due to mutationson the X chromosome (X-linked mental retardation) (3). Thedisease is associated with the expansion of CGG trinucleotiderepeats at the 50-untranslated region of the FMR1 gene ata fragile site of the X chromosome (FRAXA). Affected indi-viduals have O200 repeats, which results in hypermethylationof the promoter region, repressed transcription at the FMR1gene, and clinical expression of the disease. Premutationcarriers have 55e200 repeats (4e6).

The FMR1 gene was cloned and sequenced in 1991. It isabundantly expressed during early embryonic development

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in multiple tissues including brain and testes. The productof the FMR1 gene, the fragile X mental retardation protein(FMRP), is an RNA binding protein that is a component ofmessenger ribonucleoproteins (mRNPs) that regulate trans-lation and possibly RNA stability, which may have animpact on cellular mRNA levels. Inaccurate mRNA pro-cessing of some genes in these individuals may result inclinical manifestations of FXS or affect regulatory proteinswithin biochemical networks affecting the genome-widetranscription (5).

It is known that chromosomal fragile sites are specificloci preferentially exhibiting gaps and breaks on metaphasechromosomes resulting from partial inhibition of DNAsynthesis. Fragile sites represent intriguing components ofthe chromosome structure. Several authors suggest thatfragile sites taken significance as regions of the genome thatare particularly sensitive to replication stress and that arefrequently rearranged in tumor cells and cancer (7), corrob-orating the proposition made in 1986 by Le Beau (8).

In 1995, Panzer et al. (9) hypothesized that trinucleotideexpansions in FRAXA would also increase its cancersusceptibility. With this presumption many authors haveexplored cancer in FXS patients; nevertheless, only a verysmall group of individuals showing such association hasbeen reported: benign testicular tumor (10), seminomaand colon adenocarcinoma (11), malignant ganglioglioma(12), acute lymphoblastic leukemia (13e14), prostatecancer, glioma, meningioma (15), nephroblastoma (16),myelodysplastic syndrome (17), lung tumor (18), colorectalcancer (19), nasopharyngeal carcinoma (20), glioblastoma(21) and hepatic tumors (22). Conversely, in an extensiveand multicenter study conducted by Schultz-Pedersenet al. (23), assessing the occurrence of cancer in a largecohort of 223 FXS patients from the Danish Cytogeneticand Cancer Registries, they found a significant decreasedrisk of cancer in these patients in comparison with thegeneral population. With this same objective, in a morerecent study carried out by Sund et al. and based on anextensive sample of a Finnish population, a diminished(although nonsignificant) cancer frequency among FXSpatients was observed (24).

With the exception of Huntington disease and Downsyndrome, no genetic diseases have been reported witha decreased risk of cancer. For patients with Down syndrome,the number of solid tumors is actually significantly lower;however, the risk for leukemia is increased (25e27).

Recently, Siu and Jin (28) reported that stimulation ofcyclic adenosine monophosphate (cAMP) may either inhibitor selectively promote development of tumors depending onthe cell type. It is also known that cAMP and the responseelement binding protein (CREB) are actively involved inFMR1 transcriptional activity (29). However, it is probablethat cAMP or CREB alone may not be sufficient to inducecancer without simultaneous activation of other oncogenesor inactivation of tumor suppressor genes (30,31).

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Finally, upregulation of the Ras signaling pathway,a common cause of cancer, has recently been associatedin FXS patients (32). On the other hand, aberrant Rassignaling prevents spine maturation (33) and is linked todevelopmental disorders with facial dysmorphism (34),which are some of the most prominent clinical features inFXS (32).

Abnormal translation processing of a number of genes inFXS patients may explain not only its phenotype andclinical manifestations but the malfunction of metabolicregulatory networks. Therefore, it is reasonable to assumethat FXS patients may exhibit a differential expression ofsome proteins, particularly those related to regulation oftumor suppressor genes and/or oncogenes, which wouldexplain the decreased cancer frequency in these patients.

In order to examine such a diminished incidence ofcancer in FXS patients, we evaluate the expression of onco-genes and tumor suppressor genes using microarrayanalysis and confirmed the differential expression usingreal-time quantitative reverse transcription polymerasechain reaction.

EDMaterials and Methods

To establish the expression of oncogenes and tumorsuppressor genes in FXS patients, a comparison withnormal males was carried out.

Subjects

Ten male patients without a familial history of cancer andcarrying a complete mutation of the FMR1 gene werecompared with a control group comprised of 10 similarlyaged males (�3 years) with first-degree consanguinity.Control subjects were without malignancies or mental retar-dation and demonstrated a normal result from the molecularstudy for the FMR1 gene. FXS patients were clinicallydiagnosed at the Pediatric Hospital of the Centro MedicoNacional de Occidente, Mexican Institute of Social Secu-rity (IMSS), Guadalajara, Mexico. The study was approvedby the local institutional review board and consent proce-dures were followed accordingly (Register: 2005e108).For inclusion as patients or controls, all subjects weremolecularly analyzed for establishing the number of CGGrepeats and the methylation status of the FMR1 gene.

DNA Modification and PCR Amplification

For both groups, DNA was extracted from peripheral bloodlymphocytes using standard methods (35). DNA modifica-tion using sodium bisulfite treatment was achieved as re-ported by Panagopoulos et al. (36). PCR amplification ofthe repetitive sequence CGG was performed with primersFR526R (GGG AGT TTG TTT TTG AGA GGT GGG)and FR754F (CAA CCT CAA TCA AAC ACT CAA

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3Reduced expression of Wnt7a in Fragile X syndrome patients

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CTC CA), which were designed for amplifying unmethy-lated sequences on the antisense strand. The CpG island,located adjacent to the promoter of the FMR1 gene, wasamplified using primers FR611R (CGT CGT CGC GTTGTC GTA C) and FR690F (AAC GAC GAA CCG ACGACG). In this case, primers were designed for amplifyingonly methylated sequences. Individuals carrying a fullmutation showed methylation of this region and, conse-quently, inactivation of the FMR1 gene.

RNA Isolation and cDNA Preparation

Total RNA from peripheral blood was obtained frompatients and controls using the Pax gene blood RNA SystemKit (cat. #762134, Qiagen, Hilden, Germany) and thenfluorescently labeled with dUTP-Cy3 and dUTP-Cy5 byretrotranscription.

Microarrays

Slides were elaborated using the Human Genome Microar-ray library (MWG Biotech H10K_DB) including 10,000genes and open reading frames. Target preparation, hybrid-ization, and initial data collection were done according tothe Physiology Laboratory of the Universidad NacionalAutonoma de Mexico URL: (http://microarrays.ifc.unam.mx). Total RNA (5 mg) from patients and controls waslabeled with anchored oligo (dT) primer and incubatedwith the first-strand reaction kit component at 70�C for10 min. The second-strand reaction components were thenadded, followed by an additional 3 h incubation at 46�C andincorporation of Alexa 555 for FXS patients and Alexa 647for controls (SuperScript Plus Direct cDNA Labeling KitNucleotide Module (Invitrogen, Carlsbad, CA). cDNAwas then concentrated and purified (QIAquick PCR Purifi-cation Kit, Qiagen). The hybridization reaction was in-jected into a hybridization chamber containing the humanmicroarray and incubated with shaking (1500 rpm) for 16h at 42�C. Microarray slides were washed four times ina washing buffer followed by a final rinse in 0.1 SSC/0.05% SDS and dried by centrifugation. Two independenthybridizations (both with dye swapping) were performed,completing four hybridizations per experimental condition.

DNA Microarray Analysis

Spot detection, mean signals, mean local background inten-sities, image segmentation, and signal quantification weredetermined using the Array-Pro Analyzer 4.0 software formicroarray images (Media Cybernetics, L.P., Silver Spring,MD). Analysis of microarray data was performed with thegenArise software developed in the Computing Unit of theCellular Physiology Institute at the Universidad NacionalAutonoma de Mexico URL:(http://www.ifc.unam.mx/genarise/). This software identifies differentially expressedgenes by calculating an intensity-dependent z-score. It uses

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a sliding window algorithm to calculate the mean and stan-dard deviation (SD) within a window surrounding each datapoint and defines a z-score where z measures the number ofSDs that a data point is from the mean.

zi 5 ½Ri$meanðRÞ�=sdðRÞ

where zi is the z-score for each element, mean(R) is themean log ratio, Ri is the log ratio for each element, andsd(R) is the standard deviation of the log ratio. With thiscriterion, the elements in all experiments with a z-scoreof O2 SDs were considered significantly differentially ex-pressed genes.

We later used the Encyclopedia KEGG (Kanehisa Labo-ratories, Bioinformatics Center of Kyoto University and theHuman Genome Center of the University of Tokyo, http://www.genome.jp/kegg/) to examine the database ofgenetic and molecular pathways as previously describedin similar studies (37). KEGG is a database of biologicalsystems consisting of genetic building blocks of genesand proteins (KEGG GENES), chemical building blocksof both endogenous and exogenous substances (KEGGLIGAND), molecular wiring diagrams of interaction andreaction networks (KEGG PATHWAY), and hierarchiesand relationships of various biological objects (KEGGBRITE). KEGG provides a reference knowledge base forlinking genomes to biological systems and also to environ-ments by the processes of PATHWAY mapping and BRITEmapping.

Quantitative Real-time PCR

Cancer-related genes identified as significantly differen-tially expressed by microarray analysis were analyzed byquantitative real-time PCR (RT-PCR); each quantitativePCR was performed in duplicate. cDNA synthesis was per-formed using 5 mg of total RNA primed with oligo(dT)using the SuperScript III First-Strand Synthesis Systemfor RT-PCR (cat. #18080e051 Invitrogen) according tothe manufacturer’s recommendations. PCR primers weredesigned using the Oligo v6 software. Gene sequences wereobtained from the GenBank Nucleotide Database of theNCBI website. Primer pairs were as follows: 1) for Wnt7a,forward primer: 50-CAA AGA GAA GCA AGG CCA GTACCA-30, reverse primer: 50-GTA GCC CAG CTC CCGAAA CTG T-30; 2) for PPP2R5C, forward primer: 50-ACA GAG CAT CAT AAT GGC ATA G-30 and reverseprimer: 50-CAT TAC TTC TTT TGG ACT GTG AGT-03);for L32 ribosomal protein, forward primer: 50-GCA TTGACA ACA GGG TTC GTA G-30, reverse primer: 50-ATTTAA ACA GAA AAC GTG CAC A-30. ConventionalPCR reactions were performed using Taq DNA polymerase(cat. #11146173001, Roche Applied Science, Mannheim,Germany) and deoxynucleoside triphosphates (cat.#1969064, Roche Applied Science) in a PX2 ThermalCycler (Thermo Electron Corporation, Pittsburgh, PA).

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Table 1. Upregulated transcripts in fragile X syndrome patients

Gene Genbank number Chromosome Number of patients Description

PPP2R5C NM_601645 3p21 6 Protein phosphatase 2, regulatory subunit B (B56) gamma

ARMCX2 NM_300363 Xq21.33 5 Alex 2 protein

GUCY2D NM_600179 17p13.1 5 Guanylate cyclase 2D

IGLL1 NM_146770 22q11e21 5 Inmunoglobulin lambda-like polypeptide

RASGRF1 NM_606600 15q24 5 RAS protein-specific guanine nucleotide releasing factor 1

RRBP1 NM_601418 20p12ep11.2 5 Ribosome binding protein 1

SAS NM_605202 9 5 Sialic acid synthase

VIPR2 NM_601970 7q36.3 5 Vasoactive intestinal peptide receptor 2

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All reactions were done in a final volume of 25 ml and 60�Cof annealing temperature for 40 cycles. PCR products wereresolved in 1.5% agarose gels containing 0.1 mg/mlethidium bromide (Sigma Aldrich, Munich, Germany) visu-alized under UV light and documented with a DigiDoc-ItSystem (UVP, Upland, CA).

RT-PCR reactions were achieved using the LightCycler-FastStart DNA MasterPLUS SYBR Green I Kit (cat.#03515885001, Roche Applied Science) in a LightCycler1.5 System (Roche Diagnostics GmbH, Mannheim,Germany). PCR amplification conditions were 95�C for10 sec, annealing temperature of 60�C for 10 sec and exten-sion time of 15 sec at 72�C for 45 cycles. Melting curveswere done by temperature increases of 0.1�C/sec for60�C to 98�C. Efficiency and error of the reaction werecalculated by using a standard curve with four serial dilu-tion points of a cDNA mixture. Relative-quantificationanalysis of gene expression was done with the LightCyclerSoftware v4 using the expression of L32 ribosomal proteinas constitutive reference gene. The evaluation of relativedifferences of PCR products among patients and controlswas carried out using the DDCP method. The mean valueof the control group was adjusted to 1 and the expressionvalues for each patient are presented as percentage withregard to the control group.

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Statistical Analysis

Statistical difference between groups was evaluated by theStudent t-test; p ! 0.05 was considered statisticallysignificant.

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A human genome microarray containing 10,000 probes wasused to analyze gene expression in peripheral blood fromFXS patients and normal males. The following criteria wereused to define genes with measurable levels of expression:(1) a minimum signal intensity of 100 in at least one of thetwo probes, (2) signal-to-background ratios O2.3 for bothprobes, and (3) signal size O40% of the spotting area forboth probes. Signal intensities detected in the RNA samples

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late the mean, SD and gene differential expression valuesfor all genes in each FXS patient using the genArise soft-ware. Genes up- and downregulated in at least two SDswere analyzed.

FMR1 gene expression analyzed by microarray wasreduced or absent in all FXS patients. In comparison, expres-sion of this gene in the pool of males from the control groupshowed expression close to the median values.

Only 27 genes showed statistically significant increasedexpression (O2 SD) in FXS patients with regard to controls(Suppl. Table 3). Of these, only eight genes had this signif-icant increased expression in five or more patients (Table1). In particular, the ARMCX2 and PPP2R5C genes, whichwere found upregulated in FXS patients, are also related totumor-suppressor genes (38e41).

Likewise, 65 genes showed a statistically significantdiminished expression in FXS patients in regard to controls(Suppl. Table 4), but only 23 genes showed this statisticaldifference in at least five individuals (Table 2). The mostrelevant finding was the downregulation of the WNT7Agene, which has been related to a number of oncogenicprocesses (42). The database of genetic and molecularsignaling pathways of the genes found in the context ofKEGG biological pathways shows the importance of theWNT7A and PPP2RC5 genes within the canonic b-cateninpathway (Figure 1).

With these findings, we intentionally searched for theexpression of target genes of the b-catenin pathway. Unfortu-nately, not all target genes for this pathway were included inthe library of genes used in this study; nevertheless, c-Myc,c-Jun, cyclin-D and PPARd genes showed a moderatelyreduced expression (1.5 SD) in patients with regard to normalsubjects.

In order to validate the most interesting observations(upregulation of PPP2RC5 and downregulation ofWNT7A), a quantitative real-time PCR procedure usingintercalation of SYBR-Green was done. For this approach,only one specific fragment should be obtained from theamplification process as depicted in (Figure 2a). Tempera-ture melting point analysis of the PCR showed one peakthat represents the amplification of only one product.Additionally, conventional PCR analysis confirmed the

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Table 2. Downregulated transcripts found in fragile X syndrome patients

Gene Genbank number Chromosome Number of patients Description

CADPS NM_604667 3 7 Calcium-dependent activator protein

HRH3 NM_604525 7 Histamine receptor 3

TXNIP NM_606599 1q21 7 Thioredoxin-intereacting oxidative stress mediator

C5ORF6 NM_609372 5q31 6 Open reading frame 6

TMSB10 NM_188399 Y 6 Thymosin beta 10

AQP6 NM_601383 12q13 5 Aquaporin 6

ATP5G1 NM_603192 17 5 ATP synthase, Hþ transporting mitochondrial complex

B2M NM_109700 15q21eq22 5 Beta 2 microglobulin

EBAF NM_601877 1q41.1 5 Endometrial bleeding-associated factor

ZNF313 5 Zinc finger protein, highly expressed in testicular tissues

GMEB2 NM_607451 20 5 Glucocorticoid modulatory element binding protein 2

HHEX NM_604420 10q24 5 Hematopoietically expressed Homeobox

IL11RA NM_600939 9p13 5 Interleukin 11 receptor alpha

ITM2B NM_603904 13q14 5 Integral membrane protein 2B

KCND3 NM_605411 1p13.3ep13.2 5 Potassium channel shal-related subfamily member 3

LCP1 NM_153430 13q14eq14.3 5 Lymphocyte cytosolic protein 1

LRRC5 5

MSN NM_309845 Xq11.2eq12 5 Moesin

ROCK1 NM_601702 5 Rho-associated transcriptional activator

RP42 NM_605905 6q16 5 Expressed in human embryos

TBXA2R NM_188070 19p13.3 5 Platelet aggregation stimulator

TMSB4Y NM_400017 Y 5 Thymosin beta 4

Wnt7a NM_601570 3p25 5 Related to oncogenes

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5Reduced expression of Wnt7a in Fragile X syndrome patients

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specificity of our assay. To calculate the efficiency and errorof the PCR reaction, amplification of serial dilutions ofa cDNA mix were done, obtaining an error of 0.000451and an efficiency of 1.838 (Figure 2b). To avoid variationsin the genetic expression related to RNA extraction andcDNA synthesis, we used the same RNA samples as formicroarrays. Only nine FXS patient samples showedacceptable conditions for this assay. We used these samplesto analyze the expression of WNT7A, PPP2R5C and L32ribosomal protein. This last gene was used as referencefor the relative quantification. For this analysis, values fromthe pool of controls (healthy males) were set as 1 and thenormalized ratio was calculated by pairing each patientwith the pool of controls. As can be seen in Figure 3,

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Figure 1. Schematic representation of the Wnt signaling pathway showing the p

clopedia of Genes and Genomes, KEEG).

EDrelative expression of WNT7A was lower in FXS patients

than in control individuals, with a statistically significantdifference (!0.005) (t-test). Setting the value of the indi-viduals control as 100%, WNT7A expression in FXSpatients ranged between 21 and 76% (Figure 3b).

Expression of the PPP2RC5 gene in FSX patients was notsignificant when compared with controls (data not shown).

Discussion

For almost two decades, dynamic and highly polymorphicmutations caused by trinucleotide expansion and producinghuman illnesses have been described. To date, more than

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articipation of Wnt7a and PPP2R5C (PP2A) (modified from Kyoto Ency-

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Figure 2. Efficiency and specificity of PCR amplification. (a) Temperature melting analysis shows amplification of only one specific peak with Wnt7a

primers. Corroboration of this observation was also done by running amplification products on 1.5% agarose gel obtaining a 277-bp fragment. (b) Efficiency

and error of the PCR amplifications were assessed using standard curves generated by decreasing amounts of cDNAs (1:1, 1:2, 1:4 and 1:8, respectively)

obtaining an adequate linearity in all cases.

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one dozen of these kinds of diseases are known and, amongthem, FXS is the most important and common inheritablecause of mental retardation, which is associated with thecytogenetic expression of a fragile site on Xq27.3(FRAXA). Chromosomal instability syndromes arediseases presenting chromosomal breakpoints and typicallyhave been related to human malignancies; hence, FXSpatients constitute an interesting focus of attention forcancer research. A number of tumors occurring in patientswith this condition have been sporadically reported world-wide (10e22); nevertheless, extensive population studiescarried out in Denmark (23) and Finland (24) reported thatthe incidence of cancer in these patients is lower than in thegeneral population.

It is well known that the product of the FMR1 gene(FMRP), absent in FXS patients, is an RNA binding protein

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and an essential component of messenger ribonucleopro-teins (mRNPs) regulating RNA translation and stability.Furthermore, evidence that FMRP exhibits an inhibitoryactivity within the translation complex has been reported(43). FMRP appears to be also linked to micro-RNAs, add-ing more complexity to the role that it plays in regulatingthe RNA transport and translation (44e47). Abnormaltranslation processing of a number of genes in FXS patientsmay explain not only its phenotype and clinical manifesta-tions but the malfunction of metabolic regulatory networks.Some of the pathways in which FMRP may be involvedhave been envisaged; however, knowledge about the disrup-tion of the transcriptome and proteome that produce FXSremains to be elucidated (48).

With these antecedents, it is reasonable to assume thatFXS patients may exhibit a differential expression of some

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TFigure 3. Graphics display the median (dark lines), 25the75th percentile

(boxes) and interquartile ranges (whiskers) from our data. (a) Box plot

graphic shows the normalized ratio of WNT7A expression of each patient

with the control group setting as 1 (DD-CP). (b) Comparison between

FXS patients and healthy controls considering all data (D-CP).

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7Reduced expression of Wnt7a in Fragile X syndrome patients

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proteins, particularly those related to regulation of tumorsuppressor genes and/or oncogenes. This would explainthe decreased cancer frequency in these patients. Few geneexpression profiling studies in FXS patients have been pub-lished (49,50) but none has explored the apparent relation-ship of these patients with cancer.

Results of gene expression achieved in our laboratory bymicroarray analysis in FXS patients showed significantchanges in the expression of a number of genes whencompared with normal males (Tables 1 and 2). Some ofthese up- or downregulated genes have not demonstrateda clear biological function or are quite unknown; severalother genes show well-established roles in different tissuesbut are unrelated to the cellular cycle or oncogenic or tumorsuppressor activities. However, significant changes inexpression of some genes involved in tumorigenicprocesses were also detected. In particular, the microarrayassay showed upregulation of the PPP2R5C gene anddiminished expression of the WNT7A gene, which areknown members of oncogene and tumor suppressor fami-lies, respectively. Interestingly, both genes are involved inthe canonical b-catenin pathway. On the other hand, as ex-pected, expression of the FMR1 gene was significantlyreduced or absent in individuals from the FXS group, whichis congruent with the overmethylation observed in thepromoter regions of the FMR1 genes from all these patients.

Wnt molecules (name derived from two genes:drosophila wingless and mouse Int1) are 19 cysteine-richsecreted glycoproteins serving as extracellular signalingmolecules and play significant roles in normal and malig-nant developmental processes (51e53). Wnt proteins acton target cells via frizzled receptors (FzeR). The Wntfamily is biologically categorized into two classes: classicaland non-classical Wnts, which usually activate the canon-

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ical and noncanonical intracellular signaling pathways,respectively (53). Canonical signaling pathway leads toincrease in cytoplasmic level of b-catenin and activationof downstream signaling molecules (54). Noncanonicalsignaling pathway leads to intracellular Ca2þ flux (it acti-vates Ca2þ dependent effecter enzymes such as PKC andCamkII) or JNKs activation (jun amino-terminal kinase),which can suppress canonical signaling cascade (55,56).Recently, Wnts have drawn attention as a set of factorsoperating in embryonic development, growth, regulationof adult tissues and cancer formation (57).

The Wnt7a molecule can bind to different transmembranereceptors Frizzled. When it binds to Fzd 9 or 10, it is associ-ated with noncanonical signaling (52,58); when it binds toFzd 2, 5 or 7, it is associated with canonical b-cateninsignaling (51,52). In the fascinating canonical b-cateninsignaling pathway (Figure 1), Wnt7a, through the differenttransmembrane receptors Frizzled (2, 5 or 7), give rise to acti-vation of the cytoplasmic disheveled (Dsh) protein, whichparticipates in the b-catenin degradation complex (adenoma-tous polyposis coli gene product, glycogen synthase kinase-3b and axin). In the absence of Wnt signaling, b-catenin isphosphorylated by GSK-3b, which leads to its rapiddegradation by the ubiquitin pathway. In response to Wntsignals, b-catenin is no longer targeted for degradation, andthis protein is accumulated in the cytoplasm. Later, stabilizedb-catenin molecules translocate into the nucleus where theybind with members of the transcription factors family asT-cell factor (Tcf) and lymphoid enhancer factor (Lef) andactivate the transcription of target genes of the Wnt signalingpathway (c-Myc, c-Jun, cyclin-D, PPARd, fra-1).

The precise biochemical mechanism by which theb-catenin degradation complex is regulated is not yet known.Alterations in protein phosphorylation states are likely centralto this regulation because all elements of the b-catenindegradation complex are phosphoproteins. A number ofprotein kinases have been shown to influence Wnt/b-cateninsignaling including GSK3b, protein kinase C and caseinkinase I and II. The protein phosphatase involved in thissignaling pathway is the catalytic subunit B56 (PPP2R5C)of the protein phosphatase serine/threonine 2A (PP2A), whichexerts a positive role on Wnt/b-catenin pathway activity inmammalian cells and Xenopus embryo explants (41,59).

Quantitative RT-PCR achieved to validate the downregu-lation of the Wnt7a gene as a candidate to explain thedecreased cancer risk in patients with FXS showed a realreduction of the signal intensity in FXS males in compar-ison with healthy males. However, quantitative RT-PCRresults for the PPP2R5C gene were not able to validatethe upregulation observed with the microarray assays inmost FXS patients.

Diminished expression of the WNT7A gene, a mainregulator of the canonical b-catenin pathway, certainlyconstitutes a profound change for both the activity of thispathway as well as for the accomplishment of other

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multiple interconnected biochemical pathways. Given thecrucial importance of the WNT7A/b-catenin pathway intothe general cellular function, downregulation of WNT7Ashould be totally or partially counteracted by the actionof other involved regulators. Therefore, it is explicable thattarget genes of the WNT7A/b-catenin pathway (c-Myc,c-Jun, cyclin-D and PPARd) show in this study onlya moderate downregulation in most patients in comparisonto controls. b-catenin target genes can be not wholly absentin any cell; hence, downregulation of this pathway causedby the poor stimulation of a scant Wnt7a protein couldnot completely block the expression of the c-Myc, c-Junor cyclin-D genes but limits a possible overproduction ofthese genes. Such a moderate downregulation would evadepotential abnormalities in the cellular cycle and, conse-quently, oncogenic process.

Although the overwhelming majority of publishedreports invoke a transforming role for Wnt proteins,a limited number of studies have also invoked a tumorsuppressor role for specific Wnt proteins (58,59). Recently,several authors have remarked on this duality about thefunction of Wnt7a. Upregulation of WNT7A in endometrialtissue induced by progestogens is a finding that mayaccount for the antineoplastic effect of these hormones onthe endometrium. Also, analysis of uterine leiomyomasdemonstrated a frequent reduction in WNT7A expressionrelative to the adjacent myometria (52,60). Downregulationof the WNT7A gene has been related to the activation of thecanonical signaling pathway (52). Memarian et al. (2007)found that downexpression of WNT7A is not associatedwith suppressive effects, but probably the noncanonicalsignaling pathway can be activated later and the effectsare highly associated with the cellular receptor context.Additionally, different expression patterns of molecules ofWnt in various types of cancer may be due to the heteroge-neity of cancers and also to different mechanisms involvedin tumorigenesis (53).

Among the upregulated transcripts, the ARMCX2 genewas also present. This gene has mainly been related tothe family of proteins named ALEX and involved in devel-opment, maintenance and tissue integrity. Tumorsuppressor activity has been also proposed for this gene(38,39). Quantitative confirmation of its increased expres-sion and which molecular pathway is involved constitutean interesting task in this group of patients.

Patients with FXS represent a very interesting naturalbiological model to study a large group of molecular andmetabolic pathways that may be affected by the absenceof FMRP. Although FXS patients present with no excessivenumber of phenotypic abnormalities, a number of biochem-ical alterations affecting mainly the central nervous systemprobably are occurring. These features are caused by theabsence of FMRP that has a central role for nuclear andcytoplasmic activities, especially those related with thetranscription processes.

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In conclusion, these results suggest that the diminishedexpression of the WNT7A gene and its consequent downre-gulation of the b-catenin pathway may be related toa supposed protection of FXS patients to present cancer.

F

AcknowledgmentsThe authors thank the technical assistance of Dr. Jorge Ramırez,Lorena Chavez Gonzalez, Simon Guzman Leon and Jose SantillanTorres with the microarray preparation and the GenArise software.This study was supported by the Health Research Coordination ofthe Mexican Institute of Social Security.

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References1. Howard-Peebles P, Maddalena A, Black S, et al. Population screening

for fragile X syndrome. Lancet 1993;341:770.

2. Warren S, Sherman S. The fragile X syndrome. In: Scriver CR,

Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited

Disease. 7th edn. New York: McGraw-Hill;2001. pp. 1257e1289.

3. Lisik M, Sieron A. X-linked mental retardation. Med Sci Monit 2008;

14:RA221e229.

4. Bell M, Hirts M, Nakahori Y, et al. Physical mapping across the fragile

X: hypermethylation and clinical expression of the fragile X

syndrome. Cell 1991;64:861e866.

5. Nolin S, Lewis F, Ye L, et al. Familial transmission of the FMR1 CGG

repeat. Am J Hum Genet 1996;59:1252e1261.

6. Bourgeois J, Coffey S, Rivera S, et al. Fragile X permutation

disorders-expanding the psychiatric perspective. J Clin Psychiatry

2009;70:852e862.

7. Durkin S, Glover T. Chromosome fragile sites. Annu Rev Genet 2007;

41:169e192.

8. Le Beau M. Chromosomal fragile sites and cancer-specific rearrange-

ments. Blood 1986;67:849e858.

9. Panzer S, Kuhl D, Caskey C. Unstable triplet repeat sequences:

a source of cancer mutations. Stem Cells 1995;13:146e157.

10. Del Pozo B, Millard P. Demonstration of the fra(X) in lymphocytes,

fibroblasts, and bone marrow in a patient with a testicular tumor.

J Med Genet 1982;20:225e227.

11. Phelan M, Stevenson R, Collins J, et al. Fragile X syndrome and

neoplasia. Am J Med Genet 1988;30:77e82.

12. Rodewald L, Miller D, Sciorra L, et al. Brief clinical report: central

nervous system neoplasm in a young man with Martin Bell

syndrome-fra (X)eXLMR. Am J Med Genet 1987;26:7e12.

13. Cunningham B, Dickerman J. Fragile X syndrome and acute lympho-

blastic leukaemia. Cancer 1988;62:2383e2386.

14. Au W, Man C, Pang A, et al. Acute lymphoblastic leukemia in a patient

with fragile X syndrome. Haematologica 2003;88. ECR13.

15. Shabtai F, Hart J, Klar D, et al. Fragile X expression in Martin-Bell

syndrome, intellectually normal individuals, and neoplasia, interpreted

by a viral hypothesis. Am J Med Genet 1988;30:697e702.

16. Drouin V, Vannier J, Moirot H, et al. Nephroblastoma and fragile X

syndrome. Arch Fr Pediatr 1992;49:477.

17. Vorts E, Levene N, Nisani R, et al. Fragile X syndrome and myelodys-

plasia discovered during pregnancy. Br J Haematol 1993;85:415e416.

18. de Graaff E, Willensem R, Zhong N, et al. Instability of the CGG

repeat and expression of the FMR1 protein in a male fragile X patient

with a lung tumor. Am J Hum Genet 1995;57:609e618.

19. Fulchinogni-Lataud M, Olchwang S, Serre J. The fragile X CGG

repeats show a marked level of instability in hereditary non-

polyposis colorectal cancer patients. Eur J Hum Genet 1997;5:89e93.

20-3-2010 2-44-29

T

ARTICLE IN PRESS

9Reduced expression of Wnt7a in Fragile X syndrome patients

895896897898899900901902903904905906907908909910911912913914915916917918919920921922923924925926927928929930931932933934935936937938939940941942943944945946947948949

950951952953954955956957958959960961962963964965966967968969970971972973974975976977978979980981982983984985986987988989990991992993994995996997998999

UNCORREC

20. Ferrari A, Meazza C, Casanova M. Nasopharyngeal carcinoma in

a boy with fragile X syndrome. Pediatr Hematol Oncol 2000;17:

597e600.

21. Kalkunte R, Macarthur D, Morton R. Glioblastoma in boy with fragile

X: an unusual case of neuroprotection. Arch Dis Child 2007;92:

795e796.

22. Wirojanan J, Kraff J, Hawkins D, et al. Two boys with fragile X

syndrome and hepatic tumors. J Pediatr Hematol Oncol 2008;30:

239e241.

23. Schultz-Pedersen S, Hasle H, Olsen H, et al. Evidence of decreased

risk of cancer in individuals with fragile X. Am J Med Genet 2001;

103:226e230.

24. Sund R, Pukkala E, Patja K. Cancer incidence among persons with

fragile X syndrome in Finland: a population based study. J Intellect

Disabil Res 2009;53:85e90.

25. Hecht F, Ramesh K, Lockwood D. A guide to fragile sites on human

chromosomes. Cancer Genet Cytogenet 1990;44:37e45.

26. Sorensen S, Fenger K, Olsen J. Significantly lower incidence of cancer

among patients with Huntington disease. Cancer 1999;86:1342e1346.

27. Hasle H, Clemmensen I, Mikkelsen M. Risk of leukaemia and solid

tumors in individuals with Down’s syndrome. Lancet 2000;355:

165e169.

28. Li X, Jin P. Macro role(s) of microRNAs in fragile X syndrome. Neu-

romolecular Med 2009;. (Epub ahead of print).

29. Siu Y, Jin D. CREBa real culprit in oncogenesis. FEBS J 2007;274:

3224e3232.

30. Al-wadei H, Schuller H. Cyclic adenosine monophosphate-dependent

cell type specific modulation of mitogenic signaling by retinoids in

normal and neoplastic lung cells. Cancer Detect Prev 2006;30:

403e411.

31. Smith K, Nicholls R, Reines D. The gene encoding the fragile X RNA

binding protein is controlled by nuclear respiratory factor 2 and CREB

family of transcription factors. Nucleic Acids Res 2006;34:

1205e1215.

32. Hu H, Qin Y, Bochorishvili G. Ras signaling mechanisms underlaying

impaired GluR1 dependent plasticity associated with fragile X

syndrome. J Neurosci 2008;28:7847e7862.

33. Govek E, Newey S, Van A. The role of the Rho GTPases in neuronal

development. Genes Dev 2005;19:1e49.

34. Schubbert S, Bollag G, Shannon K. Deregulated Ras signaling in

developmental disorders: new tricks for an old dog. Curr Opin Genet

Dev 2007;17:15e22.

35. Miller S, Dykes D, Polesky H. A simple salting out procedure for ex-

tracting DNA from human nucleated cells. Nucleic Acids Res 1988;

16:1214.

36. Panagopoulos I, Lassen C, Kristoffersson U, et al. Methylation PCR

approach for detection of fragile X syndrome. Hum Mut 1999;14:

71e79.

37. Kanehisa M, Goto S, Hattori M, et al. From genomics to chemical

genomics: new developments in KEGG. Nucleic Acids Res 2006;34:

D354e357.

38. Kurochkin I, Yonemitsu N, Funahashi S, et al. ALEX1, a novel human

armadillo repeat protein that is expressed differentially in normal

tissues and carcinomas. Biochem Biophys Res Commun 2001;280:

340e347.

39. Smith C, McClive P, Sinclair A. Temporal and spatial expression

profile of the novel armadillo-related gene, Alex 2, during testicular

differentiation in the mouse embryo. Dev Dyn 2005;233:188e193.

40. Deichmann M, Polychronidis M, Nackers J, et al. The protein

phosphatase 2A subunit Bgamma gene is identified to be differentially

ARCMED1450_proof �

EDPROOF

expressed in malignant melanomas by subtractive suppression hybrid-

ization. Melanoma Res 2001;6:577e585.

41. Li H, Cai X, Shouse G, et al. A specific PP2A regulatory subunit, B56

gamma mediates DNA damage-induced dephosphorylation of p53 at

Thr55. EMBO J 2007;26:402e411.

42. Kirikoshi H, Katoh M. Expression of Wnt7a in human normal tissues

and cancer, and regulation of Wnt7a and Wnt7b in human cancer. Int J

Cancer 2002;21:895e900.

43. Laggerbauer B, Ostareck D, Keidel E, et al. Evidence that fragile X

mental retardation protein is a negative regulator of translation.

Hum Mol Genet 2001;10:329e338.

44. Jin P, Zarnescu D, Ceman S, et al. Biochemical and genetic interaction

between the Fragile X mental retardation protein and the microRNA’s

pathway. Nat Neurosci 2004;7:113e117.

45. Jin P, Alisch R, Warren S. RNA and microRNA’s in fragile X mental

retardation protein. Nat Cell Biol 2004;6:1048e1053.

46. O’Donnell W, Warren S. A decade of molecular studies of fragile X

syndrome. Annu Rev Neurosci 2002;25:315e338.

47. Bagni C, Greenough W. From mRNP trafficking to spine dysmorpho-

genesis: the roots of fragile X syndrome. Nat Rev Neurosci 2005;6:

376e387.

48. Li Y, Lin L, Jin P. The microRNA pathway and Fragile X mental retar-

dation protein. Biochim Biophys Acta 2008;1779:702e705.

49. Brown V, Jin P, Ceman S, et al. Microarray identification of FMRP

associated brain mRNAs and altered mRNA translational profiles in

Fragile X Syndrome. Cell 2001;107:477e487.

50. Bittel D, Kibiryeva N, Butler M. Whole genome microarray analysis

of gene expression in subjects with fragile X syndrome. Genet Med

2007;9:464e472.

51. Caricasole A, Ferraro T, Iacovelli L, et al. Functional characterization

of WNT7A signaling in PC12 cells. J Biol Chem 2003;39:

37024e37031.

52. Carmon K, Loose D. Secreted frizzled-related protein 4 regulates two

Wnt7a signaling pathways and inhibits proliferation in endometrial

cancer cells. Mol Cancer Res 2008;6:1017e1028.

53. Memarian A, Tehrani J, Vossough P, et al. Expression profile of Wnt

molecules in leukemic cells from Iranian patients with acute myelo-

blastic leukemia. Iran J Immunol 2007;3:145e154.

54. Khan N, Bendall L. Role of Wnt signaling in normal and malignant

hematopoiesis. Histol Histopathol 2006;21:761e774.

55. Oishi I, Suzuki H, Onishi N, et al. The receptor tyrosine kinase Ror2 is

involved in non-canonical Wnt5a/JNK signalling pathway. Genes

Cells 2003;8:645e654.

56. Mikels A, Nusse R. Purified Wnt5a protein activates or inhibits b-cat-

enin-TCF signaling depending on receptor context. PLoS Biol 2006;4:

570e582.

57. Dosen G, Tenstad E, Nygren MK, et al. Wnt expression and canonical

Wnt signaling in human bone marrow B lymphopoiesis. BMC Immu-

nol 2006;29:13e29.

58. Winn R, Marek L, Han SY, et al. Restoration of Wnt-7a expression

reverses non-small cell lung cancer cellular transformation through

frizzled-9-mediated growth inhibition and promotion of cell differen-

tiation. J Biol Chem 2005;20:19625e19634.

59. Lindberg D, Akerstrom G, Westin G. Mutational analyses of Wnt7a

and HDAC11 as candidate tumour suppressor genes in sporadic malig-

nant pancreatic endocrine tumours. Clin Endocrinol 2007;66:

110e114.

60. Gaetje R, Holtrich U, Karn T, et al. Characterization of Wnt7a expres-

sion in human endometrium and endometriotic lesions. Fertil Steril

2007;6:1534e1540.

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Suppl Table 3. Complete list of upregulated transcripts in fragile X syndrome patients

Gene Genbank number Chromosome Description

PPP2R5C NM_601645 3p21 Protein phosphatase 2, regulatory subunit B (B56) gamma

ARMCX2 NM_300363 Xq21.33 Alex 2 protein

GUCY2D NM_600179 17p13.1 Guanylate cyclase 2D

IGLL1 NM_146770 22q11e21 Inmunoglobulin lambda-like polypeptide

RASGRF1 NM_606600 15q24 RAS protein-specific guanine nucleotide releasing factor 1

RRBP1 NM_601418 20p12ep11.2 Ribosome binding protein 1

SAS NM_605202 9 Sialic acid synthase

VIPR2 NM_601970 7q36.3 Vasoactive intestinal peptide receptor 2

ZNF167

AFTIPHILIN Component of clathrin machinery in neurons

CTSF NM_603539 11q13.1 Cathepsin F

ETS1 NM_164720 11q23.3 Transcription factor

HBQ1 NM_142240 16pterep13.3 Hemoglobin theta-1-locus

IPO8 NM_605600 12p11.2 Importin 8, Ran binding protein

PRDX1 NM_176763 1p34.1 Tumor suppressor proliferation associated gene A

CRYGB NM_123670 2q33-q35 Lens protein

CTAG1B NM_300156 Xq28 Cancer/testis antigen 1B

EEF1G NM_130593 Eukaryotic translation elongation factor 1 gamma

FBLN5 NM_604585 14q32.1 Promote vascular development and remodeling

FKBP4 NM_600611 T-cell FK505 binding protein

FLJ21308

GDAP1L1

HBD NM_142000 11p15.5 Hemoglobin-Delta -Locus

BAX NM_600040 19q13.3q13.4 BCL2-associated protein

MSH6 NM_600678 2p16 Mismatch-binding protein

TSG101 NM_601387 11p15.2ep15.1 Tumor susceptibility gene 101

WT1 NM_607102 11p13 Wilms tumor 1 gene

Suppl Table 4. Complete list of downregulated transcripts found in fragile X syndrome patients

Gene Genbank number Chromosome Description

CADPS NM_604667 3p21.1 Calcium-dependent activator protein

HRH3 NM_604525 Histamine receptor 3

TXNIP NM_606599 1q21 Thioredoxin-intereacting oxidative stress mediator

C5ORF6 NM_609372 5q31 Open reading frame 6

TMSB10 NM_188399 Y Thymosin beta 10

AQP6 NM_601383 12q13 Aquaporin 6

ATP5G1 NM_603192 17 ATP synthase, Hþ transporting mitochondrial complex

B2M NM_109700 15q21eq22 Beta 2 microglobulin

EBAF NM_601877 1q41.1 Endometrial bleeding-associated factor

ZNF313 NM_612451 20q13 Zinc finger protein, highly expressed in testicular tissues.

GMEB2 NM_607451 20q13.33 Glucocorticoid modulatory element binding protein 2

HHEX NM_604420 10q24 Hematopoietically expressed Homeobox

IL11RA NM_600939 9p13 Interleukin 11 receptor alpha

ITM2B NM_603904 13q14 Integral membrane protein 2B

KCND3 NM_605411 1p13.3ep13.2 Potassium channel shal-related subfamily member 3

LCP1 NM_153430 13q14eq14.3 Lymphocyte cytosolic protein 1

LRRC5 NM_612890 1p22.2 Leucine rich repeat containing protein 8D

MSN NM_309845 Xq11.2eq12 Moesin

ROCK1 NM_601702 18q11.1 Rho-associated transcriptional activator

RP42 NM_605905 6q16 Expressed in human embryos

TBXA2R NM_188070 19p13.3 Platelet aggregation stimulator

TMSB4Y NM_400017 Yq11.221 Thymosin beta 4

WNT7A NM_601570 3p25 Related to oncogenes

ARTN NM_603886 Neuroblastina

ASAH2 NM_611202 N-acylsphingosine amidohydrolase 2

ATP6V1D NM_609398 14q24 ATPase, transporting, lysosomal VI subunit D

(continued on next page)

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Suppl Table 4 (continued )

Gene Genbank number Chromosome Description

C14ORF131

CCL3 NM_182283 17q12 Chemokine, CC motif, ligand 3

CD48 NM_ 109530 1q21.3eq22 B-cell activation marker CD48 antigen

CLTB NM_118970 4q2eq3 Clathrin light polypeptide B

COL15A1 NM_120325 9q21eq22 Collagen type XV

DREV1 NM_ 609388 Inmunoglobulin superfamily

FKBP11 NM_610571 12q13 Catalyze the folding of proline containing polypeptides

FLJ20551

FZD7 NM_603410 2q33 Frizzled receptor

GALNACT-2 NM_602274 1q41eq42 UDP-Acetyl-alpha-D- galactosa

GOSR1 NM_604026 17q11 Golgi Snap receptor complex member 1

GPR12 NM_600752 13q12 G protein coupled receptor 12

HBD

HLA-E NM_143010 6p21.3 Major histocompatibility complex class I

HS3ST3A1 NM_604057 17p12ep11.2 Heparan sulfate D-glucosaminyl 3-O sulfotransferase 3A1

IARS NM_600709 9q21 Isoleucyl-TRNA synthetase

IGFALS NM_601489 16p13.3 Insulin-like growth factor-binding protein

JAK1 NM_147795 1p31.3 Janus kinase

KIAA1036 NM_609011 Vasohibin 1

LIPELOC51234

MAP3K4 NM_602425 6q26 Mitogen-activated protein kinase kinase

Kinase 4

MAPK6 NM_602904 Mitogen-activated protein kinase 6

MBD5 NM_611472 2q23.1 Methyl-CpG binding domain protein 5

MEFV NM_608107 16p13 Familial Mediterranean fever gene

NAG18

NDUFV1 NM_161015 11q13 NADH-ubiquinone oxidoreductase Flavoprotein 1

NPM3 NM_606456 10q24eq26 Nucleophosmin/nucleoplasmin family, member 3

P2RY2 NM_600041 11q13.5 Purinergic receptor P2Y, G-protein coupled

PRDX1 NM_176763 1p34.1 Proliferation-associated gene

PRG1 NM_177040 10q22.1 Proteoglycan 1

RAD9A NM_603761 11q13.1eq13.2 Protein related to DNA replication

RDH5 NM_601617 12q13eq14 Retinol dehydrogenase

RENBP NM_312420 Xq28 Renin-binding protein

SERPING1 NM_606860 11q11eq13.1 Complement component 1 inhibitor

TNFRSF10B NM_603612 8p22ep21 Tumor necrosis factor receptor superfamily member 10B

TULP3 NM_604730 12p13 Tubby-like protein 3

UGT2B4 NM_600067 4q13 Uridine diphospate glycosyltranferase 2 family

WARP NM_611901 1p36.3 Von Willebrand factor A domain-containing protein 1

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9.e2Reduced expression of Wnt7a in Fragile X syndrome patients

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