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©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 11 (1): 467-483 (2012) Gene expression profiles in the cerebellum of transgenic mice over expressing the human FMR1 gene with CGG repeats in the normal range J.J. Fernández 1 , R. Martínez 1 , E. Andújar 2 , M. Pérez-Alegre 3 , A. Costa 1 , V. Bonilla-Henao 1 , F. Sobrino 1 , C.Ó. Pintado 4 and E. Pintado 1 1 Department of Medical Biochemistry and Molecular Biology, University Hospital Virgen Macarena, University of Seville, Spain 2 Genomics Unit, Andalusian Molecular Biology and Regenerative Medicine Centre, CSIC, Seville, Spain 3 Genomics Unit, Andalusian Molecular Biology and Regenerative Medicine Centre, Seville, Spain 4 Breeding and Research Animal Center, University of Seville, Seville, Spain Corresponding author: E. Pintado E-mail: [email protected] Genet. Mol. Res. 11 (1): 467-483 (2012) Received July 20, 2011 Accepted October 17, 2011 Published March 1, 2012 DOI http://dx.doi.org/10.4238/2012.March.1.4 ABSTRACT. Modifications in the GABA pathway are considered to be responsible for motor alterations in animal models for fragile X-associated tremor ataxia syndrome. We analyzed the expression profile in the cerebellum in a transgenic mouse model that over expresses the human FMR1 gene with CGG repeats in the normal range. We used the “GeneChip Mouse Gene 1.0 ST Array” from Affymetrix analyzing 28,853 well-described and -characterized genes. Based on data from the comparative analysis of the expression profile, we detected a significant gradient with a P value <0.1 and changes in expression equal to or greater than 1.5 times compared to the control mouse genes. There were significant changes in the expression of 104 genes, among which 72% had decreased and 28% had increased expression. With the exception

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©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (1): 467-483 (2012)

Gene expression profiles in the cerebellum of transgenic mice over expressing the human FMR1 gene with CGG repeats in the normal range

J.J. Fernández1, R. Martínez1, E. Andújar2, M. Pérez-Alegre3, A. Costa1, V. Bonilla-Henao1, F. Sobrino1, C.Ó. Pintado4 and E. Pintado1

1Department of Medical Biochemistry and Molecular Biology, University Hospital Virgen Macarena, University of Seville, Spain2Genomics Unit, Andalusian Molecular Biology and Regenerative Medicine Centre, CSIC, Seville, Spain3Genomics Unit, Andalusian Molecular Biology and Regenerative Medicine Centre, Seville, Spain4Breeding and Research Animal Center, University of Seville, Seville, Spain

Corresponding author: E. PintadoE-mail: [email protected]

Genet. Mol. Res. 11 (1): 467-483 (2012)Received July 20, 2011Accepted October 17, 2011Published March 1, 2012DOI http://dx.doi.org/10.4238/2012.March.1.4

ABSTRACT. Modifications in the GABA pathway are considered to be responsible for motor alterations in animal models for fragile X-associated tremor ataxia syndrome. We analyzed the expression profile in the cerebellum in a transgenic mouse model that over expresses the human FMR1 gene with CGG repeats in the normal range. We used the “GeneChip Mouse Gene 1.0 ST Array” from Affymetrix analyzing 28,853 well-described and -characterized genes. Based on data from the comparative analysis of the expression profile, we detected a significant gradient with a P value <0.1 and changes in expression equal to or greater than 1.5 times compared to the control mouse genes. There were significant changes in the expression of 104 genes, among which 72% had decreased and 28% had increased expression. With the exception

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of GabarapL2, no changes in expression of genes from the GABA pathway were observed, which may explain the absence of an altered motor phenotype in these mice. These results further support the view that toxic effects in fragile X-associated tremor ataxia syndrome are due to expansion of CGG repeats rather than increased mRNA levels, since in the transgenic mice the FMR1 mRNA levels were increased 20-100 times compared with those of control littermates.

Key words: Animal model; Microarrays; FMR1; Cerebellum; FXTAS

INTRODUCTION

Loss of expression of the FMR1 gene by increased CGG trinucleotide repeats (<200) in the 5'UTR causes the most frequent inherited form of mental retardation (fragile X syn-drome, FXS), whereas carriers of premutation alleles (55-200 CGG triplet repeats) may present a specific late-onset neurodegenerative disorder characterized by tremor, ataxia, par-kinsonism, and intellectual decline (fragile X-associated tremor ataxia syndrome, FXTAS) (Hagerman et al., 2001; Hagerman and Hagerman, 2004a; Jacquemont et al., 2007; Costa et al., 2011; Greco et al., 2011). Neurohistological studies on the brain of premutation carriers have demonstrated neuronal degeneration in the cerebellum and the presence of eosinophilic intranuclear inclusions in both neurons and astroglia (Jacquemont et al., 2003; Greco et al., 2006; Wenzel et al., 2010).

The increase in CGG repeats is parallel to an increase in FMR1 mRNA levels without significant changes in FMR1 mRNA stability (Kenneson et al., 2001; Loesch et al., 2007; Tassone et al., 2007). The knock-in mouse model generated in which the endogenous CGG repeat was replaced by a human CGG repeat in the premutation range displays biochemical, phenotypic and neuropathological characteristics of FXTAS (Willemsen et al., 2003). As in humans, the expanded CGG repeat mouse model shows elevated fmr1 mRNA levels in the brain compared with controls (Willemsen et al., 2003; Brouwer et al., 2007; Hunsaker, 2011). The elevated level of this abnormal mRNA is believed to be the cause of the neurodegenera-tive disorder. In a Drosophila model expressing a portion of the premutated human FMR1 5'UTR the repeats may cause neurodegeneration in a dosage- and repeat length-dependent manner (Jin et al., 2003). An almost normal CGG repeat of 60 triplets, when moderately ex-pressed, has little phenotype, and this same allele, when overexpressed, does lead to neurode-generation, supporting the notion that overall rCGG abundance is critical for the pathological phenotype (Jin et al., 2003). The toxicity of the FMR1 mRNA has been related to the excess recruitment of one or more RNA-binding proteins to the expanded repeats causing depletion and loss of function of these proteins (Hagerman and Hagerman, 2004b, 2007). Dysfunction in RNA metabolism has also been involved in the pathogenesis of several neurological disorders (Ginsberg et al. 1998; Gallo et al., 2005; Oostra and Willemsen, 2009; Lemmens et al., 2010).

It has been reported that overexpression of the FMR1 gene with CGG in the normal range does not rescue the fragile X phenotype in KO mouse (Bakker et al., 2000) although a reversal of sensomotor gating abnormalities in the KO mice carrying a human FMR1 trans-gene has been described (Paylor et al., 2008). However, a more detailed study of transgenic mice without CGG expansion is missing. To ascertain whether an increase in the FMR1 mes-

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Microarray analysis of the cerebellum in FMR1 transgenic mice

senger level, independently of the CGG number, may affect the expression profile in cerebel-lum we performed a microarray analysis from a transgenic mouse model that overexpresses (20-100-fold) the human FMR1 gene with CGG in the normal range.

MATERIAL AND METHODS

Animal models

The experiments were conducted in accordance with institutional guidelines and ap-proved by the Animal Ethics Committee of the University of Seville. We generated a trans-genic line that overexpresses human FMR1 with CGG in the normal range (29 repeats). An EcoRI fragment containing the human FMR1 cDNA kindly supplied by Dr. Verker (Erasmus Medical Center, Rotterdam, The Netherlands) was cloned in the EcoRI site of the expression vector pSG5. The plasmid plus the FMR1 cDNA was grown in an LB broth buffer and the construction was cut with SalI restriction enzyme. SalI generates two fragments, one of 4.56 kb corresponding to the FMR1 gene, T7 promoter, the β-globin intron and SV40 promoter and a fragment of 3.04 kb from the rest of the plasmid (Figure 1A).

The 4.56-kb fragment containing the FMR1 cDNA was extracted from 0.8% agarose gel and purified with a kit from Qiagen (Qiagen Iberia, Madrid, Spain) (Figure 1B). A solution of 2.5 ng/µL containing the purified 4.56-kb fragment was microinjected into the pronucleus of fertilized murine oocytes as previously described (Mejias et al., 2006) and we selected two founder lines. DNA was extracted from mouse tissue by the QuickExtractTM DNA Extraction Solution 1.0 (Epicentric, Biotechnologies, Madison, WI, USA) and tested by conventional PCR of the KH domains with primers between exons 7 and 11 to prevent genomic DNA am-plification as previously described (Hmadcha et al., 1998). The PCR was productive only in animals where the insert was incorporated in their genome (Figure 1C).

RNA extraction and quantitative RT-PCR

Cortex, cerebellum and liver tissues of transgenic mice and control littermates were dissected, placed immediately in TRIsure buffer and RNA was extracted as indicated by the manufacturer (Bioline, Luckenwalde, Germany). The concentration and quality of total RNA were analyzed spectrophotometrically. RNA was stored at -80°C until used. Reverse transcrip-tion (RT) reaction was performed in 40 µL with 0.5 to 1 µg total RNA, 1X PCR buffer, 5.5 mM MgCl2, 1 mM each dNTP, 5 µM random primers, 0.4 RNAse inhibitor and 2.5 U M-MLV reverse transcriptase (Promega, Madison, WI, USA).

Quantitative (fluorescence) RT-PCR was performed in an ABI Prism 7300 Real-Time PCR System (Applied Biosystems, USA). PCRs were performed in triplicate in a total volume of 25 µL containing 100 ng cDNA and the SensiMix SYBR Green PCR master mix following the conditions recommended by the manufacturer (Quatance, London, UK). Primer sequenc-es for human FMR1 were from exon 3 and exon 5, FMR201F: 5'-GCAGATTCCATTTCAT GATGTCA-3', and FMR327R: 5'-CAATTGTGACAATTTCATTGTAAGTT-3' as described by Allen et al. (2004). For internal control, we used hprt gene expression assessed by us-ing murine primers: hprtF: 5'-CACAGGACTAGAACACCTGC-3' and hprtR: 5'-GCTGGT GAAAAGGACCTCT-3' as described by Drabek et al. (1997).

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Quantitative RT-PCR amplification of c-fos was also performed in the ABI 7300 Real-time PCR System with gene-specific primers using the following sequences: fosF: 5'-CTGTCAACACACAGGACTTTT-3' and fosR: 5'-AGGAGATAGCTGCTCTACTTTG-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and was amplified using the following primers: GAPDH-F: 5'-CTTCACCACCATGGAGA AGGC-3' and GAPDH-R: 5'-GGCATGGACTGTGGTTCAT-3' as described by Janitzky et al. (2009). For all genes analyzed by quantitative RT-PCR the thermal cycle conditions consisted of initial denaturation at 95°C for 10 min followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Melting curve analysis showed a single sharp peak with the expected Tm for all samples. Determinations of cycle threshold were performed automatically by the instrument and calculations were done as described by Tassone et al. (2000a).

Figure 1. A. Scheme of the plasmid used to obtain the transgenic mice. The characteristics of the pSG5 plasmid are shown. The plasmid is cut with EcoRI where the cDNA of the human FMR1 (3.5 kb) was inserted. The construct was incorporated into competent cells and grown in a medium with ampicillin. B. Plasmid extraction and digestion with SalI. The plasmid was extracted with a kit from Quiagen and digested with the restriction enzyme SalI. Two fragments of 4.561 and 3.039 kb were obtained. The 4.561-kb fragment contains FMR1 cDNA, SV40 promoter and beta-globin intron. This fragment was separated on 0.8% agarose gel and purified. Dilutions were made for the injection in fertilized ovocites. Lane ND = plasmid non-digested; lane D = plasmid digested with SalI. C. Detection of transgenic mice. Two founder lines were obtained (lines A and B). Genotyping was performed by conventional PCR of the KH domains and yielded a 500-bp fragment. Only animals that have incorporated the FMR1 gene amplified the 500-bp fragment. Groups of both positive (+/-) and negative (-/-) mice of the same line and age were selected for the experiments. Lanes 1, 2, 3, 4, 6, 8, 10, 13, 15, 16, 17, and 18 = negative mice; lanes 5, 7, 9, 11, 12, and 14 = positive mice; lane 19 = a positive control.

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Microarray analysis of the cerebellum in FMR1 transgenic mice

Microarray analysis

For microarray experiments RNA from transgenic mice and control littermates was extracted and maintained at 80°C until used. Gene expression profile was determined by us-ing a “GeneChip Mouse Gene 1.0 ST Array” by Affymetrix platform at the Genomics Unit of CABIMER (Seville, Spain) containing 28,853 well-described and characterized genes. Quality of total RNA from mice was confirmed with Bioanalyzer® 2100 (Agilent technol-ogy). Synthesis, labeling and hybridization were performed with RNA from three independent mice of each condition following Affymetrix recommended protocols. Probe signal intensities were captured and processed with the GeneChip® Operating Software 1.4.0.036 (Affymetrix), and the resulting CEL files were reprocessed using robust multi-array average normalization (Irizarry et al., 2003). Fold change (log2) values and their P values were calculated with linear models for microarray analysis (Smyth, 2004), using the oneChannelGUI interface (Sanges et al., 2007). All statistical analyses were performed using R language and the packages freely available from the “Bioconductor Project” (http://www.bioconductor.org). With the data re-sulting from the comparative analysis of the expression profile, we established a significant grade with P value <0.1 and linear fold change in expression equal to or above 1.5 times above the control mouse. The functional annotation was analyzed using the DAVID Bioinformatics Database (http://david.abcc.ncifcrf.gov/home.jsp). The association of differentially expressed genes with genetic disorders and neurological diseases as well as hepatic diseases was identi-fied using the IPA 9.0 software (Ingenuity Systems, www.ingenuity.com) available through the PAB (The Andalusian Platform of Bioinformatics www.scbi.uma.es) from the University of Malaga.

Assessment of exploration and activity

Open-field behavior was recorded in a brightly lit 50 x 50-cm arena. Mice always started from the center of the arena and were allowed 1 min of adaptation before the 60-min recording period commenced. A computerized video-tracking system (Smart.V2.5, Panlab, Barcelona, Spain) was used to record trajectories and calculate path length and time spent in the square periphery of the arena (Van Dam, 2005).

Statistical analysis

Exploration and activity data are presented as means ± SD, with the number (N) of ex-periments indicated. The statistical analysis of the data was performed using a non-parametric test. In particular, the Mann-Whitney U-test was used to check for statistical differences in distance covered and time spent in the periphery of the arena (PT%) between the control and the transgenic group. P values smaller than 0.1 were considered to be statistically significant.

RESULTS

We used the pronuclear injection of the 4.56-kb fragment to generate a transgenic mouse model that overexpresses human FMR1 with CGG trinucleotide repeats in the normal range (29 repeats). Animals were genotyped by conventional PCR of the KH domain as in-

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dicated in Material and Methods (see Figure 1C). Two founder lines, A and B, were obtained and positive (+/-) and negative (-/-) animals of the same age were maintained to perform the experiments.

Quantitative RT-PCR showed that the relative FMR1 mRNA level in all tissues ana-lyzed from transgenic mice was much higher than in the wild type. Figure 2 shows in bar diagrams the mean of three different experiments of FMR1 mRNA levels in cerebellum, cor-tex and liver from mice at 14 weeks of age from line B. FMR1 mRNA in the cerebellum of transgenic mice was 20 times higher than in the wild type, and the expression in cortex and liver tissue of transgenic mice was even higher compared to controls (50-100 times).

Figure 2. Quantitative RT-PCR of mFMR1 in transgenic mice. Cerebellum (Cr), liver tissue (L) and cortex (Co) were dissected from transgenic and wild-type mice. RNA was isolated and reverse transcribed as indicated in Material and Methods. Real-time PCR showed that the expression of human FMR1 was 20 to 100 times (fold change) higher than the values in control littermates, which were normalized to one (C). The results are reported as means ± SD of three different experiments from line B.

Behavioral analysis of male mice at 7 and 14 months did not show statistically signifi-cant differences between transgenic mice and control littermates (Table 1). The animals did not reveal significant differences in general activity or anxiety-related behaviors in the open-field test. Similar results were obtained using female mice of 3 and 11 months (data not shown).

Wild type Transgenic

Distance PT% Distance PT%

7 months 13883 ± 5377 (6) 13.57 ± 5.7 (6) 12187 ± 2561 (6) 16.04 ± 7.43 (6)14 months 11923 ± 4777 (4) 23.2 ± 4.73 (4) 11928 ± 2770 (4) 15.79 ± 11.18 (4)

Distance covered is reported in cm and permanence time (PT%) in percent of the total time spent in the periphery of the arena. Data are reported as means ± SD. Number of animals analyzed is indicated in parentheses.

Table 1. Assessment of exploration and activity.

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Microarray analysis of the cerebellum in FMR1 transgenic mice

For microarray analysis, we used transgenic mice of 20 weeks of age and control littermates. Considering the significant grade and linear changes in expression indicated in Material and Methods, we observed changes in 75 well-described genes in which 70% are inhibited and 30% increased by FMR1 overexpression. Following the data supported by IPA 9.0 (see Material and Methods), we divided the changed genes into four categories including those related to neurological diseases and the GABAergic signaling pathway (Figure 3).

Table 2 shows a detailed description of the 35 genes corresponding to the groups of neurological diseases (blue columns in Figure 3) and genetic disorders (green columns in Fig-ure 3). A Veen diagram shows the genes shared by both groups and a subset of genes involved also in hepatic diseases.

The most affected gene is transthyretin (Ttr), a carrier of thyroxine and retinol that decreases four times compared with controls. On the other hand, Serpina3, a serine proteinase inhibitor (α-1-antichymotrypsin), is the most up-regulated gene from the neurological disease group. The data show that with the exception of the up-regulation of GabarapL2 no change in expression of genes from the GABAergic pathway was observed. We have confirmed by real-time PCR the up regulation of cfos obtained in the microarray experiments (data not shown).

Figure 3. Gene expression profile in the cerebellum from transgenic mice. Cerebellar tissue was dissected and RNA was extracted as described in Material and Methods. cDNA was obtained by reverse transcription and expression was analyzed by “GeneChip Mouse ST 1.0 Array” manufactured by Affymetrix. The diagram shows the 75 well-characterized genes that present changes equal to or above 1.5 times with respect to control littermates and with a P value <0.1. From these genes 70% were inhibited (down in the graphic) and 30% were increased (up in the graphic). Genes related to neurological diseases are represented in blue, genes related to known genetic disorder in green and, other misregulated genes in yellow. With the exception of GabarapL2 (red column) no known gene from the GABAergic pathway was altered.

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r,

mem

ber 3

pr

ogre

ssiv

e su

pran

ucle

ar p

alsy

,

co

rtico

basa

l deg

ener

atio

n,

la

te-o

nset

Alz

heim

er’s

dis

ease

,

ch

roni

c fa

tigue

synd

rom

e,

ac

ute

rena

l allo

graf

t rej

ectio

n,

Pa

rkin

son’

s dis

ease

, fro

ntot

empo

ral

dem

entia

with

par

kins

onis

m,

Hun

tingt

on’s

dis

ease

RTL1

re

trotra

nspo

son-

EN

SMU

ST00

0001

4 EN

SEM

BL

///

3

5332

6 ///

G

enet

ic d

isor

ders

N

on-in

sulin

-dep

ende

nt

like

1 90

46 //

/ EU

4349

18

Gen

Ban

k ///

38

7154

diab

etes

mel

litus

/// N

M_1

8410

9 ///

R

efSe

q ///

NR

_029

550

///

Ref

Seq

///

mm

u-m

ir-13

6 m

iRB

ase

Mic

ro

RN

A D

atab

ase

AB

CB

5 AT

P-bi

ndin

g EN

SMU

ST00

0000

3 EN

SEM

BL

///

777

06

Gen

etic

dis

orde

rs

Rhe

umat

oid

arth

ritis

, cor

onar

y ar

tery

ca

sset

te,

5515

// A

Y76

6239

G

enB

ank

///

dise

ase,

non

-Hod

gkin

’s d

isea

se,

su

bfam

ily B

///

NM

_029

961

Ref

Seq

mul

tiple

mye

lom

a,

(MD

R/T

AP)

,

ac

ute

mye

loid

leuk

emia

,

mem

ber 5

le

ukop

enia

, men

ingi

oma,

prim

ary

bilia

ry c

irrho

sis,

brai

n ca

ncer

, glio

blas

tom

a, c

ance

r

Tabl

e 2.

Con

tinue

d.

Con

tinue

d on

nex

t pag

e

Page 10: Gene expression profiles in the cerebellum of transgenic ...

476

©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (1): 467-483 (2012)

J.J. Fernández et al.Sy

mbo

l En

trez

Gen

e N

ame

Tran

scrip

t ID

So

urce

dat

abas

e En

trez

Gen

e ID

C

lust

er IP

A a

ssig

nmen

t D

isea

ses

MSX

2 m

sh h

omeo

box

2 EN

SMU

ST00

0000

2 EN

SEM

BL

///

177

02

Gen

etic

dis

orde

rs

Enla

rged

par

ieta

l for

amin

a, p

arie

tal

1922

/// B

C14

1132

G

enB

ank

///

fora

min

a cl

eido

cran

ial d

yspl

asia

,

///

NM

_013

601

Ref

Seq

panc

reat

ic c

ance

r, pa

ncre

atic

car

cino

ma,

no

nsyn

drom

ic c

rani

osyn

osto

sis

PRLR

pr

olac

tin re

cept

or

ENSM

UST

0000

012

ENSE

MB

L ///

1

9116

G

enet

ic d

isor

ders

, A

myo

troph

ic la

tera

l scl

eros

is,

4470

/// B

C09

6586

G

enB

ank

///

N

euro

logi

cal d

isea

ses

Cro

hn’s

dis

ease

, can

cer,

endo

met

ritis

,

///

NM

_011

169

Ref

Seq

endo

met

rial h

yper

plas

ia, m

ucin

ous o

varia

n

ca

ncer

, muc

inou

s ova

rian

carc

inom

a, c

lear

-cel

l

ov

aria

n ca

rcin

oma,

ova

rian

carc

inom

a,

en

dom

etrio

id c

arci

nom

a, u

terin

e ca

ncer

,

en

dom

etria

l car

cino

ma,

ova

rian

canc

er,

sero

us o

varia

n ad

enoc

arci

nom

a, m

edul

lary

thyr

oid

canc

er, f

amili

al m

edul

lary

thyr

oid

canc

er, t

hyro

id c

ance

r, he

ad a

nd n

eck

canc

er,

neop

lasi

a, tu

mor

igen

esis

, hyp

erpr

olac

tinem

ia,

insu

lin re

sist

ance

, hyp

ergl

ycem

ia, o

besi

ty,

hypo

insu

linem

ia, h

yper

lept

inem

ia,

hypo

glyc

emia

, exp

erim

enta

lly in

duce

d

ad

enom

yosi

s, hy

poca

lcem

iaN

R4A

1 nu

clea

r rec

epto

r EN

SMU

ST00

0000

2 EN

SEM

BL

///

153

70

Gen

etic

dis

orde

rs,

Hun

tingt

on’s

dis

ease

, prim

ary

bilia

ry

subf

amily

4,

3779

/// B

C00

4770

G

enB

ank

///

N

euro

logi

cal d

isea

ses,

ci

rrho

sis,

poly

artic

ular

juve

nile

rheu

mat

oid

gr

oup

A, m

embe

r 1

/// N

M_0

1044

4 R

efSe

q

Hep

atic

syst

em d

isea

se

arth

ritis

, end

omet

riosi

s, pr

imar

y sc

lero

sing

chol

angi

tis, o

steo

arth

ritis

, der

mat

itis,

ENPP

2 ec

tonu

cleo

tide

ENSM

UST

0000

004

ENSE

MB

L ///

1

8606

G

enet

ic d

isor

ders

, B

ipol

ar d

isor

der,

Cro

hn’s

dis

ease

, rhe

umat

oid

py

roph

osph

atas

e/

1591

/// B

C05

8759

G

enB

ank

///

N

euro

logi

cal d

isea

ses,

arth

ritis

, met

asta

sis,

Ret

t syn

drom

e,

phos

phod

iest

eras

e 2

/// N

M_0

1574

4 R

efSe

q

Hep

atic

syst

em d

isea

se

tube

rcul

oid

lepr

osy,

chr

onic

fatig

ue sy

ndro

me,

met

asta

tic c

olor

ecta

l can

cer,

hepa

toce

llula

r

ca

rcin

oma,

live

r neo

plas

ia, l

iver

can

cer,

canc

erle

t-7

mic

roR

NA

let-7

b N

R_0

2972

8 ///

R

efSe

q ///

38

7246

G

enet

ic d

isor

ders

, C

ance

r, br

east

can

cer,

liver

neo

plas

ia,

mm

u-le

t-7c-

1 m

iRB

ase

Mic

ro

N

euro

logi

cal d

isea

ses,

liver

can

cer,

hepa

toce

llula

r car

cino

ma,

R

NA

Dat

abas

e

Hep

atic

syst

em d

isea

se

infe

ctio

n by

Cry

ptos

pori

dium

par

vum

,

A

lzhe

imer

’s d

isea

se, n

onob

stru

ctiv

e

az

oosp

erm

ia, s

chiz

ophr

enia

, lun

g ca

ncer

,

lu

ng sq

uam

ous c

ell c

arci

nom

a, li

mb

gird

le

m

uscu

lar d

ystro

phy

type

2B

, mel

anom

a

m

etas

tase

s, sq

uam

ous-

cell

carc

inom

a, li

mb

gird

le m

uscu

lar d

ystro

phy

type

2A

,

fa

cios

capu

lohu

mer

al m

uscu

lar d

ystro

phy,

nem

alin

e m

yopa

thy,

mel

anom

a, M

iyos

hi

m

yopa

thy,

leio

myo

mat

osis

, lei

omyo

ma,

Dow

n’s s

yndr

ome,

pro

stat

e ca

ncer

, pro

stat

ic

ca

rcin

oma,

bre

ast c

arci

nom

a, m

etas

tasi

s

Tabl

e 2.

Con

tinue

d.

Con

tinue

d on

nex

t pag

e

Page 11: Gene expression profiles in the cerebellum of transgenic ...

477

©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (1): 467-483 (2012)

Microarray analysis of the cerebellum in FMR1 transgenic miceTa

ble

2. C

ontin

ued.

Con

tinue

d on

nex

t pag

e

Sym

bol

Entre

z G

ene

Nam

e Tr

ansc

ript I

D

Sour

ce d

atab

ase

Entre

z G

ene

ID

Clu

ster

IPA

ass

ignm

ent

Dis

ease

s

KC

NE2

po

tass

ium

vol

tage

- EN

SMU

ST00

0000

4 EN

SEM

BL

///

2461

33

Gen

etic

dis

orde

rs

Ang

ina

pect

oris

, acu

te m

yoca

rdia

l inf

arct

ion,

ga

ted

chan

nel,

7383

/// B

C02

2699

G

enB

ank

///

cong

estiv

e he

art f

ailu

re, l

ung

canc

er,

Is

k-re

late

d fa

mily

, ///

NM

_134

110

Ref

Seq

atria

l fibr

illat

ion,

ven

tricu

lar fi

brill

atio

n,

mem

ber 2

ve

ntric

ular

tach

ycar

dia,

ven

tricu

lar

arrh

ythm

ia, a

trial

fibr

illat

ion,

fam

ilial

,

lo

ng q

t syn

drom

e va

riant

3, h

ypoc

hlor

hydr

ia,

achl

orhy

dria

, hyp

erga

strin

emia

, hyp

erpl

asia

CLD

N1

clau

din

1 EN

SMU

ST00

0000

2 EN

SEM

BL

///

127

37

Gen

etic

dis

orde

rs

Cor

onar

y ar

tery

dis

ease

, NIS

CH

synd

rom

e,

31

54 //

/ BC

0020

03

Gen

Ban

k ///

br

east

can

cer,

colo

n ca

ncer

/// N

M_0

1667

4 R

efSe

qD

USP

1 du

al sp

ecifi

city

EN

SMU

ST00

0000

2 EN

SEM

BL

///

192

52

Gen

etic

dis

orde

rs,

Hyp

ertro

phy,

flu,

end

omet

riosi

s, pr

osta

tic

phos

phat

ase

1 50

25 //

/ BC

0069

67

Gen

Ban

k ///

Neu

rolo

gica

l dis

ease

s, ca

rcin

oma,

leio

myo

mat

osis

, lei

omyo

ma,

/// N

M_0

1364

2 R

efSe

q

Hep

atic

syst

em d

isea

se

expe

rimen

tally

indu

ced

diab

etes

,

co

llage

n-in

duce

d ar

thrit

is, w

eigh

t los

s,

at

opic

der

mat

itis,

psor

iasi

s, br

east

can

cer,

rheu

mat

oid

arth

ritis

, pro

stat

e ca

ncer

,

ex

perim

enta

l aut

oim

mun

e en

ceph

alom

yelit

isH

SPA

1A/

heat

shoc

k EN

SMU

ST00

0000

8 EN

SEM

BL

///

1937

40

Gen

etic

dis

orde

rs,

Hun

tingt

on’s

dis

ease

, ins

ulin

-dep

ende

ntH

SPA

1B

70-k

Da

7328

/// B

C05

4782

G

enB

ank

///

N

euro

logi

cal d

isea

ses

diab

etes

mel

litus

, can

cer,

pr

otei

n 1A

///

NM

_010

479

Ref

Seq

Alz

heim

er’s

dis

ease

, hep

atoc

ellu

lar

carc

inom

a, sc

hizo

phre

nia,

live

r neo

plas

ia,

liver

can

cer,

obes

ity, n

euro

dege

nera

tion,

blad

der c

ance

r, bl

adde

r car

cino

ma,

expe

rimen

tal c

oliti

s, ac

idos

is,

adre

nole

ukod

ystro

phy,

pol

yarti

cula

r juv

enile

rheu

mat

oid

arth

ritis

, end

omet

riosi

s,

am

yotro

phic

late

ral s

cler

osis

, wei

ght l

oss,

tum

orig

enes

is, h

yper

plas

ia, P

arki

nson

’s

di

seas

e, is

chem

ia, t

rem

or, h

yper

troph

yH

RH

4 hi

stam

ine

ENSM

UST

0000

004

ENSE

MB

L ///

22

5192

G

enet

ic d

isor

ders

, Vo

miti

ng, m

etas

tatic

bre

ast c

ance

r, ur

ticar

ia,

re

cept

or H

4 16

76 //

/ AF3

5885

9 G

enB

ank

///

N

euro

logi

cal d

isea

ses

mot

ion

sick

ness

, nau

sea,

cor

onar

y ar

tery

/// N

M_1

5308

7 R

efSe

q

di

seas

e, b

reas

t car

cino

ma

TTR

tra

nsth

yret

in

ENSM

UST

0000

007

ENSE

MB

L ///

2

2139

G

enet

ic d

isor

ders

, A

lzhe

imer

’s d

isea

se, a

myl

oido

sis,

5312

/// D

8907

6 ///

G

enB

ank

///

N

euro

logi

cal d

isea

ses,

ne

urol

ogic

al d

isor

der,

seni

le sy

stem

ic

N

M_0

1369

7 R

efSe

q

Hep

atic

syst

em d

isea

se

amyl

oido

sis,

syst

emic

reac

tive

amyl

oido

sis,

fam

ilial

am

yloi

dotic

pol

yneu

ropa

thy,

hepa

tic sy

stem

dis

orde

r, ca

ncer

, lun

g ca

ncer

,

br

onch

iolo

-alv

eola

r ade

noca

rcin

oma,

first

-ons

et p

aran

oid

schi

zoph

reni

a,

co

ld th

yroi

d no

dule

, maj

or d

epre

ssio

n,

ex

perim

enta

lly in

duce

d di

abet

es

Page 12: Gene expression profiles in the cerebellum of transgenic ...

478

©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (1): 467-483 (2012)

J.J. Fernández et al.Ta

ble

2. C

ontin

ued.

Sym

bol

Entre

z G

ene

Nam

e Tr

ansc

ript I

D

Sour

ce d

atab

ase

Entre

z G

ene

ID

Clu

ster

IPA

ass

ignm

ent

Dis

ease

s

PLU

NC

pa

late

, lun

g an

d EN

SMU

ST00

0000

2 EN

SEM

BL

///

1884

3 G

enet

ic d

isor

ders

N

on-s

mal

l cel

l lun

g ca

ncer

, non

-sm

all c

ell

na

sal e

pith

eliu

m

8985

/// B

C05

4375

G

enB

ank

///

lung

car

cino

ma,

lung

can

cer,

lung

as

soci

ated

///

NC

_013

697

Ref

Seq

aden

ocar

cino

ma,

can

cer

LBP

lipop

olys

acch

arid

e EN

SMU

ST00

0000

1 EN

SEM

BL

///

1680

3 G

enet

ic d

isor

ders

, N

on-in

sulin

-dep

ende

nt d

iabe

tes m

ellit

us,

bi

ndin

g pr

otei

n 61

68 //

/ BC

0047

95

Gen

Ban

k ///

Hep

atic

syst

em d

isea

se

coro

nary

arte

ry d

isea

se, h

yper

tens

ion,

/// N

M_0

0848

9 R

efSe

q

pr

ogre

ssiv

e fa

mili

al in

trahe

patic

chol

esta

sis t

ype

1, li

ver c

ance

r, C

rohn

’s

di

seas

e, p

neum

onia

, sep

sis,

rheu

mat

oid

arth

ritis

, hep

atic

stea

tosi

s, pn

eum

ococ

cal

pneu

mon

ia, e

xper

imen

tal

colit

is, b

acte

rial p

neum

onia

NR

4A2

nucl

ear r

ecep

tor

ENSM

UST

0000

011

ENSE

MB

L ///

18

227

Gen

etic

dis

orde

rs,

Fam

ilial

Par

kins

on’s

dis

ease

, ato

pic

su

bfam

ily 4

, 26

29 //

/ BC

1377

15

Gen

Ban

k ///

Neu

rolo

gica

l dis

ease

s de

rmat

itis,

prog

ress

ive

supr

anuc

lear

gr

oup

A,

/// N

M_0

1361

3 R

efSe

q ///

pa

lsy,

pol

yarti

cula

r juv

enile

m

embe

r 2

NM

_001

1395

09

Ref

Seq

rheu

mat

oid

arth

ritis

, psy

chos

is,

T-ce

ll no

n-H

odgk

in’s

dis

ease

,

pe

riphe

ral T

-cel

l lym

phom

a,

Pa

rkin

son’

s dis

ease

, bre

ast c

ance

r,

os

teoa

rthrit

is, p

soria

sis

MEI

S2

Mei

s hom

eobo

x 2

ENSM

UST

0000

014

ENSE

MB

L ///

17

536

Gen

etic

dis

orde

rs,

Hyp

erte

nsio

n, in

sulin

-dep

ende

nt

92

17 //

/ U57

343

///

Gen

Ban

k ///

Neu

rolo

gica

l dis

ease

s di

abet

es m

ellit

us, H

untin

gton

’s d

isea

se,

NM

_010

825

///

Ref

Seq

auto

som

al d

omin

ant p

olyc

ystic

kidn

ey d

isea

seS1

00A

2 S1

00 c

alci

um

GEN

SCA

N00

0000

1 EN

SEM

BL

///

6283

24

Gen

etic

dis

orde

rs

Psor

iasi

s, se

rous

ova

rian

carc

inom

a

bind

ing

prot

ein

A2

60

41 //

/ R

efSe

q ///

pr

oces

s, se

rous

ova

rian

carc

inom

a,

X

M_0

0147

8157

///

Ref

Seq

dela

yed

hype

rsen

sitiv

e re

actio

n,

X

M_9

1061

1

liche

n pl

anus

, end

omet

rioid

car

cino

ma,

brea

st c

ance

r, at

opic

der

mat

itis,

ovar

ian

canc

er, c

lear

-cel

l ova

rian

carc

inom

a, m

ucin

ous o

varia

n ca

ncer

,

m

ucin

ous o

varia

n ca

rcin

oma,

can

cer

ND

ST4

N-d

eace

tyla

se/N

- EN

SMU

ST00

0001

4 EN

SEM

BL

///

6458

0 G

enet

ic d

isor

ders

, A

lzhe

imer

’s d

isea

se

sulfo

trans

fera

se

3461

/// A

B03

6838

G

enB

ank

///

N

euro

logi

cal d

isea

ses

(h

epar

an g

luco

sam

inyl

) 4

/// N

M_0

2256

5 R

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J.J. Fernández et al.

These results indicate that the increase in the expression of human FMR1 mRNA with CGG triplets in the normal range in mice produced mild changes in the transcriptome but did not affect the GABAergic pathway or induce the motor alterations described in the animal model of FXTAS. The possible significance of the altered gene expression profile in the trans-genic mice reported here should be further analyzed.

DISCUSSION

The involvement of the GABAergic system in both FXS and FXTAS, the two faces of the FMR1 gene, has been reported (D’Hulst et al., 2009). Expression analysis of fmr1 KO mice compared to wild type shows decreased expression of several subunits of the GABAA receptor in fragile X mouse cortex, but not in cerebellum. By contrast overexpression of sev-eral GABAA receptor subunits and proteins involved in GABA metabolism has been observed in cerebellum but not in the cortex of the mice model for FXTAS (D’Hulst et al., 2009). This is consistent with the cerebellar phenotype of FXTAS patients (D’Hulst et al., 2009) although the precise mechanistic relationship between CGG size and clinical phenotype is still unclear. It is likely that a combination of CGG repeat length and FMR1 message abundance together may define a threshold for the clinical manifestation of the disease (Jin et al., 2003; Willemsen et al., 2003; Brouwer et al., 2007). In our transgenic mice expression of human FMR1 mRNA is 20 to 100 times higher than in controls in any tissue analyzed (see Figure 2). These levels are an order of magnitude higher than the 2-6-fold elevated FMR1 mRNA levels found in

Venn diagram. Intersection of genes from different groups. The criteria used to define candidate differentially expressed genes are indicated in Material and Methods. Genes from “Other misregulted” group (yellow columns in Figure 3) are not described.

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Microarray analysis of the cerebellum in FMR1 transgenic mice

premutation carriers or in the transgenic CGG-expanded repeat mouse model (Tassone et al., 2000a; Willemsen et al., 2003).

The high expression of human FMR1 mRNA results in a differential expression pat-tern in cerebellum but only the GabarapL2 gene from the GABAergic pathway was changed. According to these results, we did not find any motor phenotype in males or females at differ-ent ages (see Table 1). These data agree with the absence of correlation between Fmr1 mRNA levels and neuropathological features found in the CGG-repeat knock-in mouse model (Brou-wer et al., 2008). This study further supports the view that gain-of-function in FXTAS arises as a result of the expanded CGG repeats rather than the abnormally increased levels of FMR1 mRNA present in a carrier of premutation alleles. Therefore, it would be of interest to know if fragile X males with unmethylayed full-mutation trinucleotide repeat expansions (Tassone et al., 2000b) show a severe form of FXTAS or an early presentation due to the very large CGG repeat expansion.

Interestingly, the two most altered genes from the group of neurological diseases, Trt and Serpina 3, found in our transgenic mice, are related to Alzheimer disease. A decrease in TRT has been associated with late onset Alzheimer disease and it is used in cerebrospinal fluid as a bio-marker (Buxbaum et al., 2008). On the other hand, up-regulation of Serpina 3 is found in Alzheimer patients (Porcellini et al., 2008). Thus, this may suggest an RNA toxicity that would work through alteration of specific genes. A subgroup of modified gene expression is related to hepatic diseases (see Venn diagram and Table 2). Since the transgenic mice reported here express extremely high FMR1 mRNA levels in liver it would be of interest to know the gene expression profile of this tissue and its correlation with a possible hepatic phenotype.

The increase in c-fos proto-oncogene observed in the microarray has been validated by quantitative RT-PCR. However, Fmr1 mRNA was not changed in the microarray analysis due to specific amplification of mouse Fmr1 without recognition of human FMR1 cDNA.

In conclusion, differential expression of genes determined by microarray analysis from transgenic mice versus wild type did not induce changes in the GABAergic system and transgenic mice did not show a cerebellar phenotype. The changes in the transcriptome may produce a non-cerebellar phenotype that should be further investigated.

ACKNOWLEDGMENTS

We would like to thank Dr. Verker for kindly supplying the human FMR1 cDNA. We also thank Dr. López-Barneo for critical reading of the manuscript. Research supported by a Grant from the Instituto de Salud Carlos III, Ministerio de Educación y Ciencia de España (#PI081332). J.J. Fernández was supported by a fellowship from the Hospital Universitario Virgen Macarena de Sevilla.

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