New polymorphisms in human MEF2C gene as potential modifierof hypertrophic cardiomyopathy

Cristina Alonso-Montes • Manuel Naves-Diaz • Jose Luis Fernandez-Martin •

Julian Rodriguez-Reguero • Cesar Moris • Eliecer Coto •

Jorge B. Cannata-Andia • Isabel Rodriguez

Received: 10 January 2012 / Accepted: 7 June 2012 / Published online: 21 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Hypertrophic cardiomyopathy is caused by

mutations in genes encoding sarcomeric proteins. Its vari-

able phenotype suggests the existence of modifier genes.

Myocyte enhancer factor (MEF) 2C could be important in

this process given its role as transcriptional regulator of

several cardiac genes. Any variant affecting MEF2C

expression and/or function may impact on hypertrophic

cardiomyopathy clinical manifestations. In this candidate

gene approach, we screened 209 Caucasian hypertrophic

cardiomyopathy patients and 313 healthy controls for

genetic variants in MEF2C gene by single-strand confor-

mation polymorphism analysis and direct sequencing.

Functional analyses were performed with transient trans-

fections of luciferase reporter constructions. Three new

variants in non-coding exon 1 were found both in patients

and controls with similar frequencies. One-way ANOVA

analyses showed a greater left ventricular outflow tract

obstruction (p = 0.011) in patients with 10C?10C geno-

type of the c.-450C(8_10) variant. Moreover, one patient

was heterozygous for two rare variants simultaneously.

This patient presented thicker left ventricular wall than her

relatives carrying the same sarcomeric mutation. In vitro

assays additionally showed a slightly increased transcrip-

tional activity for both rare MEF2C alleles. In conclusion,

our data suggest that 15 bp-deletion and C-insertion in the

50UTR region of MEF2C could affect hypertrophic car-

diomyopathy, potentially by affecting expression of

MEF2C and therefore, the expression of their target cardiac

proteins that are implicated in the hypertrophic process.

Keywords Hypertrophic cardiomyopathy �Modifier genes � Genetic polymorphism � MEF2C

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-012-1740-7) contains supplementarymaterial, which is available to authorized users.

C. Alonso-Montes � M. Naves-Diaz � J. L. Fernandez-Martin �J. B. Cannata-Andia � I. Rodriguez

Bone and Mineral Research Unit, Hospital Universitario Central

de Asturias, Oviedo, Spain

C. Alonso-Montes � M. Naves-Diaz � J. L. Fernandez-Martin �E. Coto � J. B. Cannata-Andia � I. Rodriguez

Instituto Reina Sofıa de Investigacion Nefrologica, Oviedo,

Spain

M. Naves-Diaz � J. L. Fernandez-Martin � E. Coto �J. B. Cannata-Andia � I. Rodriguez

Red Tematica de Investigacion Cooperativa REDinREN,

Oviedo, Spain

J. Rodriguez-Reguero � C. Moris

Area del Corazon-Fundacion Asturcor, Hospital Universitario

Central de Asturias, Oviedo, Spain

E. Coto

Unidad de Genetica, Hospital Universitario Central de Asturias,

Oviedo, Spain

E. Coto � J. B. Cannata-Andia

Department of Medicine, University of Oviedo, Oviedo, Spain

I. Rodriguez (&)

Servicio de Metabolismo Oseo y Mineral, Hospital Universitario

Central de Asturias, Edif. Polivalente A, 2a pl., C/Julian

Claverıa, s/n, 33006 Oviedo, Spain

e-mail: irodriguez@hca.es

123

Mol Biol Rep (2012) 39:8777–8785

DOI 10.1007/s11033-012-1740-7

Introduction

Hypertrophic cardiomyopathy is characterized by an

increased ventricular wall thickness or mass in the absence

of loading conditions sufficient to cause the observed

abnormality [1]. Microscopically, it exhibits myocyte dis-

array and interstitial myocardial fibrosis [2]. The prevalence

of hypertrophic cardiomyopathy has been estimated in

approximately 1 in 500 in the general population [3], and is

frequently familial, transmitted as an autosomal dominant

trait. Most of these familial cases are caused by mutations in

one of at least 13 genes that encode sarcomeric proteins [4].

However, in all the large cohorts so far reported, a signifi-

cant number of patients did not have identified sarcomeric

mutations, suggesting that other genes could be implicated

in this disease. In addition, wide phenotypic heterogeneity

is also found, even among mutation carriers in the same

family, suggesting that some modifier factors (inherited and

acquired) are implicated in this interindividual variability

[5] modulating the phenotypic expression of the disease.

Several cardiac transcription factors regulate cardiac gene

expression and are also involved in cardiac hypertrophy in

response to several stresses to the adult heart [6]. Among

others, members of the myocyte enhancer factor (MEF) 2

family bind to the promoters of a number of cardiac genes

that are upregulated in the hypertrophied myocardium.

MEF2 is composed by four members in vertebrates (MEF2A

to D) expressed in the precursors of the three muscle lineages

[7] and they have a critical role in mouse embryogenesis. In

this way, Mef2C null mice have altered cardiac gene

expression and die during early embryonic development with

arrested heart tube morphogenesis [8]. Moreover, MEF2

transcription factors also play prominent roles in the regu-

lation of cardiac hypertrophy and remodelling [9, 10] being

its activity upregulated by prohypertrophic stimulus such as

insulin-like growth factor-1 [11], calcineurin [10], and sar-

comere gene mutations [12]. In addition, overexpression of

Mef2A or C in cultured cardiomyocytes induces sarcomere

disorganization and elongation of cardiomyocytes [9].

The modifier genes involved in hypertrophic cardiomyop-

athy and the significance of their effects are still not well

defined in humans. In the present study, a cohort of hypertro-

phic cardiomyopathy patients was screened for variations in the

human MEF2C gene. Functional in vitro analyses of these

variants were also performed to know its effect on gene activity.

Materials and methods

Patients

A total of 209 unrelated patients with hypertrophic car-

diomyopathy were recruited at the Cardiology Department

of Hospital Universitario Central de Asturias (HUCA,

Oviedo). All of them were Caucasian from the same region

of the North of Spain (Asturias, 1 million of inhabitants).

The diagnosis was performed following the American

College of Cardiology/European Society of Cardiology

(ACC/ESC) guidelines [13], using as an inclusion criteria a

left ventricular wall thickness [13 mm measured by

echocardiography, not secondary to other cardiac diseases

capable of producing left ventricular hypertrophy, such as

hypertension, valve disease, and myocardial infarction.

Most of these patients had previously been studied and all

have been screened for mutations in all exonic regions and

splice sites of the most frequently mutated sarcomeric

genes MYH7, MYBPC3, TNNT2, and a-tropomyosin

(TPM1) [14]. The main clinical and anthropometric char-

acteristics and treatments of the patients are summarized in

Table 1.

The control group consisted of 313 healthy Cauca-

sian individuals from the same region, 76 % men, aged

20–75 years (mean age 43.8 ± 12.5), and recruited through

Table 1 Clinical and echocardiographic characteristics and treatment

of the patients with hypertrophic cardiomyopathy included in the

study

Men/women (% men) 141/68 (67)

Age (years) 51 ± 18

Age of onset (years) 47 ± 18

BMI (kg/m2) 26 ± 6

Family history, n (%)

Hypertrophic cardiomyopathy 66 (31.5)

Sudden cardiac death 34 (16)

Clinical symptoms, n (%)

Angina 60 (29)

Syncope 37 (18)

Dyspnea 119 (57)

NYHA index

Class I–II 98 (82)

Class III–IV 21 (18)

Echocardiographic data

Interventricular septum (mm) 20 ± 5.6

LVOTO, n (%) 88 (42)

LVOT gradient (mmHg) 51 ± 38

Treatment, n (%)

Pharmacologic 146 (70)

Pacemaker 8 (4)

Cardiac defibrillator 8 (4)

Sarcomeric mutation, n (%) 52 (25)

Quantitative variables are shown as mean ± standard deviation.

Family history denotes at least a first-degree relative with hypertro-

phic cardiomyopathy and/or sudden cardiac death before age 50

BMI body mass index, NYHA New York Heart Association functional

class, LVOTO left ventricular outflow tract obstruction

8778 Mol Biol Rep (2012) 39:8777–8785

123

the Blood Bank and the Department of Cardiology at

HUCA. Although these controls were apparently healthy

and did not have suffered cardiac diseases, not all of them

were echocardiographically evaluated.

All individuals gave their informed consent for the

study, and this was approved by the local Ethics Com-

mittee of Clinical Investigation.

Genotyping

Genomic DNA was extracted from blood samples as pre-

viously described [15]. Coding exons, their flanking

introns, and 50 untranslated region (UTR) of the human

MEF2C gene were amplified using polymerase chain

reaction (PCR) as previously described [16]. Primers were

designed to anneal in the flanking introns on the basis of

the genomic sequence (GenBank accession numbers

NT_006713.15 and NM_002397.3). Exons 1 and 11 were

divided in two fragments because of its great size. PCR

amplicons were then analyzed by single strand conforma-

tion polymorphism (SSCP) in 12 % polyacrylamide gels, at

room temperature, as previously reported [16]. Electro-

phoretic patterns were visualized after silver staining of the

gels. PCR fragments showing different SSCP electropho-

retic patterns were sequenced on both strands using an

ABI310 system, with BigDye chemistry (Applied Biosys-

tems, Foster City, CA, USA) to determine the nucleotide

change responsible for the variation.

In silico sequence analysis

Identification of the possible transcription factor binding

motifs affected by the polymorphisms was carried out by

using the software MatInspector from Genomatix (http://

www.genomatix.de/) with the default settings (matrix sim-

ilarity 0.75). The Mfold server (http://mfold.rna.albany.edu/)

was used to evaluate the most thermodynamically stable

form of the mRNA, calculating free energies and secondary

structures for each variant using the default settings.

Plasmid constructions

A PCR fragment, from -939 to ?531 bp (1,470 bp) rela-

tive to the transcriptional start site of human MEF2C gene,

was generated by PCR using Accuzyme DNA polymerase,

(Bioline, London, UK) and the following primers: Fw-50-CTC CAA CCT CTC AGG GTG A-30 and Rv-50-TCC

TCG AGA AAT ATC AGG GG-30. As template we used

genomic DNA from the patient with two MEF2C variants

(15del/10C) and as wild type (wt) we used the DNA from a

control individual with the reference sequence. PCR

products were cloned with the Zero Blunt PCR Cloning kit

(Invitrogen, Paisley, UK), confirmed by sequencing, and

the three clones of interest were subcloned in the Kpn I and

Nhe I sites of pGL3 Basic vector (Promega Corp, Madison,

WI, USA) containing a firefly luciferase reporter gene.

Cell culture and transient transfection assays

To compare the activity of different alleles of MEF2C

promoter in driving expression, transient transfection

experiments and reporter assays were performed in two

different cell lines. Transient transfection assays were done

in human embryonic kidney (HEK) 293 and rat vascular

smooth muscle (A7r5) cells, obtained from the European

Collection of Cell Cultures (ECACC). Both cell lines were

cultured in DMEM/F12 (Lonza, Basel, Switzerland) sup-

plemented with 10 % fetal bovine serum and 1 % peni-

cillin/streptomycin, (Biochrom AG, Berlin, Germany).

Cells were seeded on 24-well plates at a concentration of

50,000 cells/well for HEK293 or 40,000 cells/well for A7r5

cells, and cultured overnight. Transient transfection of

luciferase reporter plasmids was performed using Fugene

HD (Promega) according to the manufacturer’s protocol.

The Renilla luciferase reporter plasmid pRL-TK (Promega)

was co-transfected as an internal control for transfection

efficiency. Briefly, cells were exposed to a transfection

mixture containing 0.8 lg of pGL3 constructs and 0.15 lg

of pRL-TK, and a DNA:Fugene HD ratio 3:1. Cells were

harvested 48 h after transfection, then rinsed one with 1 X

PBS and lysed in 100 ll Passive Lysis Buffer (Promega).

For luciferase assays, 5 ll of cell lysates were processed

using Dual-Luciferase Reporter Assay System (Promega)

and measured in an FB12 luminometer (Berthold, Pforz-

heim, Germany). MEF2C gene promoter activity was

expressed as a ratio of the activities of firefly (pGL3 Basic)

to Renilla luciferase (pRL-TK). For each construct, three

independent transfections were performed in duplicate. The

promoter activity was expressed as mean ± standard

deviation (SD).

Statistical analyses

Chi-square test, or Fisher’s exact test when necessary, were

used to compare frequencies of the different alleles in

patients and controls as well as to determine the Hardy–

Weinberg equilibrium. Continuous variables were reported

as mean ± SD and Kolmogorov–Smirnov test for nor-

mality was performed. Differences in normally distributed

variables were analyzed using one-way ANOVA and in

non-normally distributed variables by Kruskal–Wallis

analysis. Multivariate linear regression was used to adjust

for clinical and demographic characteristics. Comparison

of transcriptional activity of the different alleles in transient

transfection assays was performed using an unpaired Stu-

dent’s t test. A p value \ 0.05 was considered statistically

Mol Biol Rep (2012) 39:8777–8785 8779

123

significant. All statistical analyses were carried out by

Statistical Package for the Social Sciences (SPSS) ver-

sion17.0 for Windows.

Results

MEF2C genetic variation

No genetic variants were found in the whole coding region

of MEF2C, but three variants not previously described

were found in the non-coding exon 1 both in patients and

controls. These variants were named according to the

recommendations for the description of sequence variants

from the Human Genome Variation Society [17]: two

variations of the number of tandem repeats (VNTR) [c.-

506CCT(4_6) and c.-450C(8_10)] and a 15 bp deletion (c.-

445_-431del). The first VNTR consists in a series of 4 to 6

CCT triplet repetitions, being the most frequent 5 CCT

(99 % against 0.8 % for 6 CCT and 0.2 % for 4 CCT). The

second VNTR consists in a series of 8–10C nucleotides,

being the 9C allele the more frequent in our population

(75.6 % against 22.8 % for 10C and 1.6 % for 8C). There

were no statistical differences in neither allele nor genotype

frequencies between patients and controls for both poly-

morphisms, even adjusting for demographic and/or clinical

characteristics (Table 2). The deletion was of a nearly

perfect tandem duplication of the 15 following nucleotides

in the sequence (CCCCTCGCGCGCGCT). This deletion

appears both in patients and controls, but with no signifi-

cant differences in frequencies.

Due to the low frequencies of c.-506CCT(4_6) and c.-

445_-431del polymorphisms, only differences for the main

demographic, clinical and echocardiographic characteris-

tics between the most common genotypes in c.-450C(8_10)

polymorphism were analyzed (Table 3). A higher mean left

ventricular outflow tract obstruction (LVOTO) at rest was

found among patients who were homozygous for the 10C

allele (p = 0.02), an association that is maintained even

after adjusting for sex and age (p = 0.002).

We found a unique patient, carrying both MEF2C rare

alleles simultaneously (15del and 10C alleles). She is a

woman with severe hypertrophic cardiomyopathy (septum

of 32 mm, LVOT pressure gradient of 46 mmHg, and

dilation of left atrium of 49 mm) diagnosed at the age of

41. She carried the Asp175Asn mutation in the sarcomeric

TPM1 gene, the same mutation that is present in her

mother, brother and son, who were asymptomatic, with

mild hypertrophy (septum of 17, 20 and 27 mm, respec-

tively), without LVOTO nor dilation of left atrium, and

only carried the 15del allele of MEF2C (Fig. 1). This fact

proves that both variants are not in the same chromosome

or, at least, not under linkage disequilibrium.

In vitro transcriptional activity

The prediction of the effect of these variants in the sec-

ondary structure of the 50UTR of the mRNA was analyzed

with the Mfold program, showing that the 10C allele did

not change secondary structure or free energy compared to

the wt. However, the 15del allele showed a partially

Table 2 Comparison of

genotype and allele frequencies

of the three polymorphisms of

MEF2C in hypertrophic

cardiomyopathy patients and

controls

p: p valuea Fisher’s exact test

Polymorphism Genotypes alleles Patients, n (%) Controls, n (%) p

c.-506 CCT (4_6) Genotype [4]?[5] 1 (0.5) 1 (0.3) 0.94

[5]?[5] 205 (98.1) 307 (98.1)

[5]?[6] 3 (1.4) 5 (1.6)

Allele [4] 1 (0.2) 1 (0.2) 0.94

[5] 414 (99) 620 (99)

[6] 3 (0.7) 5 (0.8)

c.-450 C (8_10) Genotype [8]?[9] 9 (4.3) 4 (1.3) 0.08

[8]?[10] 1 (0.5) 3 (0.9)

[9]?[9] 111 (53.1) 191 (61)

[9]?[10] 72 (34.4) 100 (32)

[10]?[10] 16 (7.7) 15 (4.8)

Allele [8] 10 (2.4) 7 (1.1) 0.08

[9] 303 (72.5) 486 (77.7)

[10] 105 (25.1) 133 (21.2)

c.-445_-431 Del Genotype Ins/Ins 207 (99) 311 (99.4) 1.00a

Ins/Del 2 (1) 2 (0.6)

Allele Ins 416 (99.5) 624 (99.7) 1.00a

Del 2 (0.5) 2 (0.3)

8780 Mol Biol Rep (2012) 39:8777–8785

123

different secondary structure and a reduced stability com-

pared to wt (Fig. 2).

The analysis of this region looking for putative binding

sites for transcription factors revealed that both variations

could substantially modify the list of potential binding sites

in MEF2C promoter. The 10C allele could result in the loss

of a GAGA-box while the 15 bp deletion could affect the

binding of several transcription factors (MAZF, ZF5F, and

MZF1) (Fig. 3).

To study if these variations could account for a differ-

ential transcriptional regulation, a 1,470 bp fragment of the

MEF2C promoter containing the two different allelic

variants was cloned (pGLMEF2C-15del and pGLMEF2C-

10C) as well as the wild type promoter (pGLMEF2C-wt)

(see sequences in Fig. 4). Both variants were in different

molecules, confirming that they are in different chromo-

some in this individual. Both constructions were transfec-

ted into two different cell lines, individually or together,

confirming that they generate promoter activity compared

to empty vector (Fig. 5). In both cell types, and compared

with cells transfected with the wt, there was an increase in

luciferase activity for both variants transfected separately,

and this increase was slightly higher when they were

transfected together (Fig. 5). In HEK293 cells, both indi-

vidual alleles displayed a significant increase in luciferase

activities with respect to wt (p = 0.013 for 10C allele and

p = 0.045 for 15del allele), and when they were transfec-

ted together resulted in a 32 % increased of transcriptional

activity compared to wt (p \ 0.001). In A7r5 cells, the

trend was also to increase, but only 15del allele displayed a

significant increase (p = 0.034) with respect to wt.

Discussion

Polymorphisms in the regulatory regions of genes might

have a notable effect on the transcription and translation of

the corresponding gene. Different genotypes could confer

interindividual variation in the gene expression. Therefore,

genetic variations may have a considerable impact on the

susceptibility, severity, and clinical outcome of a disease.

Table 3 Comparison of clinical

and echocardiographic

characteristics of the patients

with hypertrophic

cardiomyopathy included in the

study, grouped by more frequent

genotypes of c.-450 C (8_10)

polymorphism

Quantitative variables are

shown as mean ± standard

deviation. Family history

denotes, at least, a first-degree

relative with hypertrophic

cardiomyopathy and/or sudden

cardiac death before age 50

BMI body mass index, NYHANew York Heart Association

functional class, LVOTO left

ventricular outflow tract

obstruction, p: p value

c.-450 C (8_10)

[9]?[9] [9]?[10] [10]?[10] p

Patients, n 111 72 16

Men, % 71 57 73 0.11

Age of onset (years) 46 ± 18 49 ± 18 46 ± 10 0.56

BMI (kg/m2) 26 ± 7 26 ± 6 24 ± 3 0.48

Family history, %

Hypertrophic cardiomyopathy 33 29 29 0.87

Sudden cardiac death 20 15 0 0.23

Clinical symptoms, %

Dyspnea 62 45 61.5 0.16

NYHA class: I–II 81 79 87.5 0.87

III–IV 19 21 12.5

Angina 28 29 38.5 0.72

Syncope 14.5 22 25 0.43

Echocardiographic data

Interventricular septum (mm) 21 ± 6 19 ± 4 20 ± 5 0.29

Posterior wall (mm) 15 ± 4 14 ± 3 15 ± 2 0.83

LVOTO, % 44 44 23.5 0.35

LVOT gradient (mmHg) 46 ± 36 48 ± 36 108 ± 19 0.02

Sarcomeric mutation, % 26 24 37 0.53

Fig. 1 Family tree of the index case (indicated with an arrow) carrier

of both rare variants of MEF2C promoter: 15del and 10C. S carrier of

sarcomeric mutation Asp175Asn on TPM1 gene, D carrier of the

15del allele of MEF2C gene, 10C carrier of the 10C allele, 9C carrier

of the 9C allele, NA not analyzed

Mol Biol Rep (2012) 39:8777–8785 8781

123

In fact, DNA variations in regulatory regions have been

previously linked to hypertrophic cardiomyopathy in

humans, in genes acting as potential modifiers for hyper-

trophic cardiomyopathy in patients carrying sarcomeric

mutations [18] or playing an important role in the devel-

opment of the disease [19, 20].

We investigated the occurrence of polymorphic sites in

the 50UTR, coding region, and flanking introns of MEF2C

and the distribution of their frequencies in healthy controls

and patients with hypertrophic cardiomyopathy. This is a

slightly variable gene since, in the studied regions only one

polymorphism with an appreciable heterozygosity in Cau-

casian population was found in the database dbSNP built

135 from NCBI. Our population did not show to be poly-

morphic for this variant, at least with our screening

method. By contrast, three new polymorphisms in the

untranslated exon 1 were found, although their frequencies

were not statistically different between controls and

patients. However, there was an association between c.-

450C(8_10) polymorphism and the value of LVOTO.

Therefore, our results would indicate that this polymor-

phism was not associated with the risk of developing

hypertrophic cardiomyopathy, but it could modulate the

phenotypic expression of the disease increasing the

LVOTO, that is known to be closely related to hypertrophic

cardiomyopathy course and seriousness [21, 22].

In vitro functional assays showed that some alleles of

two of the new polymorphisms, 15 bp deletion and 10C

allele of c.-450C(8_10) polymorphism, increased tran-

scriptional activity compared to wt, with a slightly higher

increase if both variants were simultaneously transfected.

Several transcriptions factors seem to lose their putative

Fig. 2 Secondary structure of

minimum free energy of the

50UTR region with the wild type

(wt) and the 15del sequence of

MEF2C. Arrows signal the

region mainly affected by the

variant

Fig. 3 Transcription factors

obtained through the

MatInspector analysis of the

wild type (wt) and 15del

sequences of MEF2C promoter.

Numbers indicate positions

referred to the sequence

NM_002397.3 from GenBank

8782 Mol Biol Rep (2012) 39:8777–8785

123

binding sites in the MEF2C gene carrying the 15del allele.

Some of them belong to the family of zinc finger proteins

that are described to negatively regulate several genes [23–

25]. Therefore, its loss could lead to the observed increase

in the gene transcription. Moreover, in silico analysis

predicted that the 15del allele would be the most efficiently

translated due to a less stable secondary structure (it has

highest DG values) than wt structure. It is proved that a

very stable structure inhibits translation by blocking the

migration of ribosomes [26]. Furthermore, the importance

of UTR in regulatory gene expression is underlined by the

finding that variations in 50UTR have already been found to

modulate translation and even to cause disease [27, 28].

MEF2 has been implicated in mediating cardiac hyper-

trophy and it was showed a dose-dependent cardiomyo-

pathic phenotype and a progressive reduction in ventricular

performance associated with MEF2C overexpression in the

heart of transgenic mice. Moreover, these mice showed

altered gene expression, including genes involved in

extracellular matrix remodelling, ion handling and metab-

olism [9] that, in addition, have been related to hypertro-

phic cardiomyopathy [29–32].

In our study population, a woman with severe hyper-

trophic cardiomyopathy was the only participant carrying

both MEF2C rare alleles, including the 313 controls. This

fact would suppose a theoretical increase in the

Fig. 4 Electropherograms

showing partial sequences of the

MEF2C 50UTR wild type

(a) and the two allelic variants

15del (b) and 10C (additional C

marked by an arrow) (c) cloned

for the reporter assays

Fig. 5 Luciferase reporter assays of MEF2C 50UTR variants. Frag-

ments from MEF2C promoter region, containing the two different

variants 10C and 15del, were cloned into pGL3-Basic vector and the

constructions transfected in HEK293 (a) and A7r5 (b) cells. Data

shown represent the mean of relative luciferase activity

(RLA) ± standard deviation from three experiments performed in

duplicate. Activity of the wild type (wt) allele was fit to 1 unit.

* p \ 0.05, ** p \ 0.001, compared with wt

Mol Biol Rep (2012) 39:8777–8785 8783

123

transcriptional activity of MEF2C gene. This woman car-

ried the Asp175Asn mutation in the sarcomeric TPM1

gene, characterized in previous studies by highly variable

left ventricular hypertrophy, although rarely above 30 mm,

and favourable prognosis [33, 34]. In fact, her relatives

carrying the same mutation were asymptomatic, presented

mild hypertrophy and they only carried the 15del allele of

MEF2C. In addition, this patient presented LVOTO, a

characteristic that is not commonly associated with the

Asp175Asn mutation [33], and is not present in any of her

relatives. Their genotype for other potential modifier genes,

as for example the renin–angiotensin–aldosterone system

genes [35], does not account in this case for those differ-

ences in the hypertrophic status, as was previously reported

[36]. Therefore, we could speculate that a theoretical

higher expression of MEF2C in this patient, due to the

presence of both functional rare variants of this gene, could

account for a higher expression of any of its target cardiac

genes [37], a fact that could imply the thicker left ven-

tricular wall also present in this patient.

Recently, in mice expressing a human hypertrophic

cardiomyopathy mutation it has been reported that the

prolonged Mef2 activation in its hearts was closely asso-

ciated with fetal gene reexpression, and contributes to

pathologic atrial remodelling, especially adjacent to

regions of fibrosis [12], a histopathologic hallmark of the

hypertrophic cardiomyopathy. Moreover, mice homozy-

gous for a null mutation of Mef2C showed a reduced

expression of several markers of cardiac differentiation.

Transcripts for ANF, cardiac a-actin, and a-myosin heavy

chain were down-regulated to background levels, and

myosin light chain expression was decreased significantly,

although was still detectable at a low level in the hearts of

the mutant mice [8].

Furthermore, depletion of Mef2C by small interfering

RNA attenuates the hypertrophic growth of mice left

ventricle in response to pressure overload, reduces hyper-

trophy in cardiomyocytes, and diminishes interstitial

fibrosis [38]. In addition, a marked sarcomeric disorgani-

zation and focal elongation was observed in cultured

cardiomyocytes that overexpress MEF2C [9].

The present study has some limitations. It is based in a

small number of patients, so replication in populations with

higher number of individuals should be necessary to con-

firm the present results. The control group includes indi-

viduals not echocardiographically evaluated to confirm the

absence of hypertrophy, although this could act underes-

timating our findings. Moreover, we have not measured

protein levels of MEF2C in plasma and/or cardiac tissue to

demonstrate whether the increase in transcriptional activity

really leads to higher protein levels.

In summary, any genetic variant that directly affect

MEF2C gene expression and/or function would be

expected to impact on the expression of its target genes. In

the present study, we identified a new deletion polymor-

phism of 15 bp and two VNTR in the regulatory 50UTR

region of the human MEF2C gene. Moreover, we showed

an in vitro functional effect of some variants of these

polymorphisms in the MEF2C promoter activity in two cell

lines. Therefore, we provide the first evidence that MEF2C

is a potential modifier gene for hypertrophic cardiomyop-

athy, a fact that could help in predicting prognosis and in

the design of more specific therapies.

Acknowledgments We thank the patients and their families that

consent to participate in this study. This work was supported by grants

from the Spanish Fondo de Investigaciones Sanitarias-Fondos FEDER

European Union (FIS 07/0659, 10/00173), and Red de Investigacion

Renal-REDinREN (RD06/0016) from Instituto de Salud Carlos III.

CAM and IR were financially supported by the Fundacion para el

Fomento en Asturias de la Investigacion Cientıfica Aplicada y la

Tecnologıa (FICYT).

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