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Genetic analysis of the Irx4 gene in hypertrophic cardiomyopathy

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Fatih Bayrak, M.D.,¹ Evrim Kömürcü - Bayrak, MSc.,² Bülent Mutlu, M.D.,³ Gökhan Kahveci, M.D.,³ Nihan Erginel - Ünaltuna, PhD.²

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Hypertrophic cardiomyopathy (HC) is inherited as a Mendelian autosomal dominant trait and is caused by more than 200 different mutations in genes, each encoding protein components of the cardiac sarcom-

ere.^[1,2] The disease is genetically and clinically hetero-

geneous. The severity and the onset age of the disease change depending on mutation types.^[3] Mutations in the known contractile protein genes are not found in one-third of the HC cases.

Molecular studies of HC patients without sar- comeric protein mutations revealed other genetic causes of cardiac hypertrophy, such as mutations in WKH UHJXODWRU\ Ƣ VXEXQLW RI $03DFWLYDWHG SURWHLQ

kinase,^[4-6] and lysosome-associated membrane pro- tein 2.^[7] Recently, mutations in the muscle LIM pro- tein gene have been proposed to cause HC.^[8]

The Irx4 protein (Iroquois family homeobox pro- tein 4) is predominantly expressed in the cardiac ventricles and is a necessary mediator of ventricular differentiation during cardiac development, including the adulthood period.^[9-13] No mutation screening has been performed on the human Irx4 gene which is localized in chromosome 5p15.3.^[10] In animal studies, the absence of the Irx4 protein caused inhibition of chamber-specific expression of myosin heavy chain genes which led to abnormal ventricular gene expres- sion, resulting in adult-onset cardiac hypertrophy.^[9,11]

Considering that the absence of genes under the con- trol of Irx4 caused cardiomyopathy in mice, we pre- sumed that it might have a similar effect in humans.

Therefore, this study was designed to investigate the relationship between possible mutations in protein coding region of the human Irx4 gene and HC.

PATIENTS AND METHODS

Study sample. The study was approved by the local ethics committee and each participant gave written informed consent after appropriate genetic counsel- ing. The study consisted of 68 patients (32 females, 36 males; mean age 49±17 years; range 17 to 74

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a detailed history, physical examination, 12-lead elec- trocardiography, and transthoracic echocardiography.

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females, 34 males; mean age 45±14 years; range 20 to 88 years) with normal transthoracic echocardiograms were also included as controls. The diagnosis of HC was based on the demonstration of a hypertrophied, nondilated left ventricle (wall thickness of at least 15 mm) by two-dimensional echocardiography, in the absence of other cardiac or systemic diseases that might produce hypertrophy of similar degree.^[14] The ECG recordings were obtained with a paper speed of 50 mm/sec at normal filtering. The QT interval was defined as the interval between the beginning of the QRS complex and the end of the T wave. Three consecutive cycles were manually measured in each of the standard 12 leads, and a mean value was calcu- lated from these three measurements. The QT interval was then corrected (QTc) using Bazett’s formula. The corrected dispersion of QT intervals was defined as the difference between the maximum and minimum of the corrected QT interval which could be measured in any of the 12 ECG leads.

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using standard procedures. For mutation screening, the Irx4 gene was subdivided into seven overlapping

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fragments including the intron-exon boundaries and WKHFRGLQJUHJLRQV3ULPHUVZHUHGHVLJQHGIURPIODQN- ing intronic sequences for all exons of the Irx4 gene *HQ%DQN DFFHVVLRQ QXPEHU $< *HQRPLF

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polymorphisms detected in Irx4 gene fragments were FRQILUPHGE\GLJHVWLRQZLWK0VS$,DQG6DF,,UHVWULF- tion enzymes, respectively.

Statistical analysis. Genotypic and allelic distri- butions were compared using the chi-square test.

Continuous variables were compared with a two- tailed t-test and expressed as means ± standard devia- tion (SD), while categorical variables were compared XVLQJWKHFKLVTXDUHWHVW$WZRWDLOHGp value of less WKDQZDVFRQVLGHUHGVWDWLVWLFDOO\VLJQLILFDQW$OO

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software (Windows, version 10.0).

RESULTS

The clinical, electrocardiographic, and echocardio- graphic characteristics of the patients with HC are shown in Table 2. Sixty-four patients had asymmetric

septal hypertrophy (the ratio of end-diastolic septal to OHIWYHQWULFXODUSRVWHULRUZDOOWKLFNQHVV•DQGIRXU

patients had apical hypertrophy. Basal left ventricular RXWIORZREVWUXFWLRQJUDGLHQW•PP+JZDVSUHV- HQW LQ  SDWLHQWV  $OO FRQWURO VXEMHFWV KDG

normal transthoracic echocardiograms.

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in the protein coding region of the Irx4 gene, includ- ing these four polymorphisms detected in our study:

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significantly between patients and controls.

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to be associated with a higher maximal left ventricle outflow tract gradient (48±53 mmHg vs 24±35 mmHg;

p=0.03), prolonged corrected QT dispersion (79±21

Table 2. Clinical and echocardiographic characteristics of patients with hypertrophic cardiomyopathy

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msec vs 63±18 msec; p=0.05), and albeit statistically insignificant, increased septal thickness (2.6±0.7 cm vs 2.2±0.5 cm; p=0.07).

DISCUSSION

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find any mutations in the Irx4 gene associated with HC.

Modifier genes are neither necessary nor sufficient to cause HC, but they have a significant effect on the severity of the disease.^[15] Recent reviews have pointed out that the influence of modifier genes and environ- mental factors on the phenotypic expression of HC limit the utility of, and drawing conclusions from genotype- phenotype correlation studies.^[3,15] On the other hand, VRPHPRGLILHUJHQHVVXFKDV$*SRO\PRUSKLVP

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Table 3. Genotype and allele frequencies for polymorphisms of the Irx4 gene in patients and controls

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an insertion/deletion polymorphism in the angiotensin-1 FRQYHUWLQJHQ]\PH$&(JHQHZHUHIRXQGWREHDVVR- ciated with the clinical phenotype of HC.^[16-18] $OWKRXJK

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approximately 20% to 25% of all HC cases,^[3] it was IRXQGWKDWWKH$*SRO\PRUSKLVPRI0\%3&

was also a genetic modifier for the development of left ventricular wall thickness in patients with HC.^[16]

In our study, no mutations were detected associat- HGZLWK+&EXWFRPSDUHGZLWKWKH*$DQG**JHQR- W\SHV WKH $$ JHQRW\SH RI $!* SRO\PRUSKLVP

was found to be associated with a higher maximal left ventricle outflow tract gradient, a prolonged corrected QT dispersion, and albeit statistically insignificant, increased septal thickness in patients with HC. Thus, WKH $!* SRO\PRUSKLVP RI WKH ,U[ JHQH PD\

have a modifier effect on septal thickness resulting in a prolonged corrected QT dispersion and higher outflow gradients, but these data need to be validated by further large scale studies.

In our study, mutation screening included the intron-exon boundaries and the coding regions of the Irx4 gene, but not other sites such as introns, and regu- ODWRU\HOHPHQWV3RVVLEOHSRO\PRUSKLVPVRUPXWDWLRQV

of these sites might be the genetic modifier in the development of cardiac hypertrophy because animal studies demonstrated that the absence of the Irx4 protein caused adult-onset cardiac hypertrophy.^[9,11]

However, this needs to be substantiated by further studies that include whole gene screening and expres- sion pattern analysis.

Our sample size may not be large enough to detect mutations in the Irx4 gene; therefore, further studies with broader patient populations are necessary. It may also be of value to analyze the Irx4 gene in patients without sarcomere gene mutations.

This is the first human study investigating the association between possible mutations in the Irx4 gene and HC. Four polymorphisms were identified ZLWKLQ WKH ,U[ JHQH 7KH $$ JHQRW\SH RI $!*

polymorphism was found to be associated with a higher maximal left ventricle outflow tract gradient and prolonged corrected QT dispersion.

Acknowledgments

This work was supported by the Research Fund of ,VWDQEXO8QLYHUVLW\3URMHFWQXPEHU

REFERENCES

1. Maron BJ, McKenna WJ, Danielson GK, Kappenberger /- .XKQ +- 6HLGPDQ &( HW DO $PHULFDQ &ROOHJH

of Cardiology/European Society of Cardiology clinical expert consensus document on hypertrophic cardiomyo- SDWK\$UHSRUWRIWKH$PHULFDQ&ROOHJHRI&DUGLRORJ\

Foundation Task Force on Clinical Expert Consensus Documents and the European Society of Cardiology

&RPPLWWHH IRU 3UDFWLFH *XLGHOLQHV - $P &ROO &DUGLRO

2003;42:1687-713.

2. Maron BJ. Hypertrophic cardiomyopathy: a systematic UHYLHZ-$0$

 0DULDQ$-5REHUWV57KHPROHFXODUJHQHWLFEDVLVIRU

hypertrophic cardiomyopathy. J Mol Cell Cardiol 2001;

33:655-70.

4. Bayrak F, Komurcu-Bayrak E, Mutlu B, Kahveci G,

%DVDUDQ < (UJLQHO8QDOWXQD 1 9HQWULFXODU SUHH[FL- tation and cardiac hypertrophy mimicking hypertro- phic cardiomyopathy in a Turkish family with a novel 35.$*PXWDWLRQ(XU-+HDUW)DLO

 %ODLU(5HGZRRG&$VKUDILDQ+2OLYHLUD0%UR[KROPH

J, Kerr B, et al. Mutations in the gamma(2) subunit of

$03DFWLYDWHG SURWHLQ NLQDVH FDXVH IDPLOLDO K\SHUWUR- phic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 2001;10:1215-20.

 $UDG 0 %HQVRQ ': 3HUH]$WD\GH $5 0F.HQQD

:-6SDUNV($.DQWHU5-HWDO&RQVWLWXWLYHO\DFWLYH

$03NLQDVHPXWDWLRQVFDXVHJO\FRJHQVWRUDJHGLVHDVH

mimicking hypertrophic cardiomyopathy. J Clin Invest 2002;109:357-62.

7. Danon MJ, Oh SJ, DiMauro S, Manaligod JR, Eastwood

$1DLGX6HWDO/\VRVRPDOJO\FRJHQVWRUDJHGLVHDVH

with normal acid maltase. Neurology 1981;31:51-7.

 *HLHU & 3HUURW $ 2]FHOLN & %LQQHU 3 &RXQVHOO '

Hoffmann K, et al. Mutations in the human muscle LIM protein gene in families with hypertrophic cardio- myopathy. Circulation 2003;107:1390-5.

9. Bao ZZ, Bruneau BG, Seidman JG, Seidman CE, Cepko CL. Regulation of chamber-specific gene expression in the developing heart by Irx4. Science 1999;283:1161-4.

10. Bruneau BG, Bao ZZ, Tanaka M, Schott JJ, Izumo S, Cepko CL, et al. Cardiac expression of the ventricle- specific homeobox gene Irx4 is modulated by Nkx2-5 and dHand. Dev Biol 2000;217:266-77.

11. Bruneau BG, Bao ZZ, Fatkin D, Xavier- Neto J, Georgakopoulos D, Maguire CT, et al.

Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression. Mol Cell Biol 2001;21:1730-6.

&KULVWRIIHOV 90 .HLMVHU $* +RXZHOLQJ $& &ORXW

'( 0RRUPDQ $) 3DWWHUQLQJ WKH HPEU\RQLF KHDUW

identification of five mouse Iroquois homeobox genes in the developing heart. Dev Biol 2000;224:263-74.

+RXZHOLQJ $& 'LOGURS 5 3HWHUV 7 0XPPHQKRII -

0RRUPDQ$)5XWKHU8HWDO*HQHDQGFOXVWHUVSHFLILF

expression of the Iroquois family members during mouse development. Mech Dev 2001;107:169-74.

.OXHV +* 6FKLIIHUV $ 0DURQ %- 3KHQRW\SLF VSHF-

trum and patterns of left ventricular hypertrophy in hypertrophic cardiomyopathy: morphologic observa- tions and significance as assessed by two-dimensional HFKRFDUGLRJUDSK\ LQ  SDWLHQWV - $P &ROO &DUGLRO

1995;26:1699-708.

0DULDQ $- 0RGLILHU JHQHV IRU K\SHUWURSKLF FDUGLR- myopathy. Curr Opin Cardiol 2002;17:242-52.

:DQJ3=RX<)X&=KRX;+XL50<%3&SRO\- morphism is a modifier for expression of cardiac hyper- trophy in patients with hypertrophic cardiomyopathy.

Biochem Biophys Res Commun 2005;329:796-9.

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genotypes and left ventricular hypertrophy in patients with hypertrophic cardiomyopathy. Circulation 1995;92:

1808-12.

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Gersh BJ, Ommen SR, et al. Gene-specific modifying HIIHFWVRISUR/9+SRO\PRUSKLVPVLQYROYLQJWKHUHQLQ

angiotensin-aldosterone system among 389 unrelated patients with hypertrophic cardiomyopathy. Eur Heart J 2005;26:2457-62.