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Genetic analysis of the Irx4 in hypertrophic cardiomyopathy

Hipertrofik kardiyomiyopatide Irx4 geni analizi

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 PATIENTS AND METHODS a Mendelian autosomal dominant trait and is caused Study sample. The study was approved by the local by more than 200 different mutations in , each ethics committee and each participant gave written encoding components of the cardiac sarcom- [1,2] informed consent after appropriate genetic counsel- ere. The disease is genetically and clinically hetero- ing. The study consisted of 68 patients (32 females, geneous. The severity and the onset age of the disease 36 males; mean age 49±17 years; range 17 to 74 change depending on mutation types.[3] Mutations in \HDUV ZLWK+&$OOWKHSDWLHQWVZHUHHYDOXDWHGZLWK the known contractile protein genes are not found in a detailed history, physical examination, 12-lead elec- one-third of the HC cases. trocardiography, and transthoracic echocardiography. Molecular studies of HC patients without sar- $JHDQGJHQGHUPDWFKHGKHDOWK\VXEMHFWV  comeric protein mutations revealed other genetic females, 34 males; mean age 45±14 years; range 20 to causes of cardiac hypertrophy, such as mutations in 88 years) with normal transthoracic echocardiograms WKH UHJXODWRU\ Ƣ VXEXQLW RI $03DFWLYDWHG SURWHLQ were also included as controls. The diagnosis of HC kinase,[4-6] and lysosome-associated membrane pro- was based on the demonstration of a hypertrophied, tein 2.[7] Recently, mutations in the muscle LIM pro- nondilated left ventricle (wall thickness of at least 15 tein gene have been proposed to cause HC.[8] mm) by two-dimensional echocardiography, in the absence of other cardiac or systemic diseases that The Irx4 protein (Iroquois family pro- might produce hypertrophy of similar degree.[14] The tein 4) is predominantly expressed in the cardiac ECG recordings were obtained with a paper speed ventricles and is a necessary mediator of ventricular of 50 mm/sec at normal filtering. The QT interval differentiation during cardiac development, including was defined as the interval between the beginning of the adulthood period.[9-13] No mutation screening has the QRS complex and the end of the T wave. Three been performed on the human Irx4 gene which is consecutive cycles were manually measured in each localized in 5p15.3.[10] In animal studies, of the standard 12 leads, and a mean value was calcu- the absence of the Irx4 protein caused inhibition of lated from these three measurements. The QT interval chamber-specific expression of myosin heavy chain was then corrected (QTc) using Bazett’s formula. The genes which led to abnormal ventricular gene expres- corrected dispersion of QT intervals was defined as sion, resulting in adult-onset cardiac hypertrophy.[9,11] the difference between the maximum and minimum Considering that the absence of genes under the con- of the corrected QT interval which could be measured trol of Irx4 caused cardiomyopathy in mice, we pre- in any of the 12 ECG leads. sumed that it might have a similar effect in humans. Therefore, this study was designed to investigate the Genetics analysis.'1$ZDVLVRODWHGIURPZKROHEORRG relationship between possible mutations in protein using standard procedures. For mutation screening, coding region of the human Irx4 gene and HC. the Irx4 gene was subdivided into seven overlapping

Table 1. Primers used for the amplification of Irx4 gene fragments and parameters of the PCR and SSCP analysis 2OLJRQXFOHRWLGHV ҋmҋ  6WUDQGa ([RQV )UDJPHQW 1XFOHRWLGH $QQHDOLQJ $&   7HPSHUDWXUH ƒ&     VL]H ES  QXPEHULQJ 7P ƒ&  $*  E 9ROWDJH5XQWLPH &&*&7*&7&$&777*77$ 6 ([RQ  ҋ875a   KUPLQ $*7&*7$**7&**$&$&7 $   ,96a &&7&7&7&7*&$*77&77 6 ([RQ  ,96a(   KUPLQ $$*7***&77**&$$$&7 $   ,96a $*$*$**&7**$*7****77* 6 ([RQ  ,96a   KU $*$$$*****&$7*$7**7** $   ,96a &$77*&$*$*&&7*7&7&&$* 6 ([RQ  ,96a   KUPLQ &$*7*$&$$&&7&&&$$&&&$ $   ,96a *7&7*$77&$&&7$&&$$&&& 6 ([RQ  ,96a   KU 77**&&7&*&$*$&&7*$ $   (a 7**$&*$**$&&7**$*$ 6 ([RQ  (a   KU &$&&&$*777&7$*$$&7**77 $   (a 7**$&$**$$&&$**$&7 6 ([RQ  (a   KU $*7*$$$$*$*7&**&*& $   ҋ875a a6LQGLFDWHVWKHIRUZDUGSULPHUZLWKWKHVHTXHQFHIURPWKHVHQVHVWUDQG$LQGLFDWHVWKHUHYHUVHSULPHUZLWKWKHVHTXHQFHIURPWKHDQWLVHQVHVWUDQG E$&$FU\ODPLGHFRQFHQWUDWLRQ$*$GGLWLRQRIJO\FHURO3&53RO\PHUDVHFKDLQUHDFWLRQ66&36LQJOHVWUDQGFRQIRUPDWLRQSRO\PRUSKLVP  7UN.DUGL\RO'HUQ$Uü

Table 2. Clinical and echocardiographic characteristics of patients with hypertrophic cardiomyopathy  Q  0HDQ“6' 5DQJH $JH \HDUV    “  0DOHJHQGHU   )DPLO\KLVWRU\ +\SHUWURSKLFFDUGLRP\RSDWK\   6XGGHQGHDWK   &OLQLFDOVWDWXV 6\PSWRPDWLF   $V\PSWRPDWLF   /HIWYHQWULFOH (QGV\VWROLFGLDPHWHU FP    “ (QGGLDVWROLFGLDPHWHU FP    “ 0D[LPDOZDOOWKLFNQHVV FP    “ (MHFWLRQIUDFWLRQ     “  /HIWDWULXPVL]H FP    “  3DWLHQWVZLWKUHVWLQJJUDGLHQW!PP+J   456GXUDWLRQ PVHF    “ 47GLVSHUVLRQ PVHF    “ &RUUHFWHG47GLVSHUVLRQ PVHF    “ fragments including the intron-exon boundaries and septal hypertrophy (the ratio of end-diastolic septal to WKHFRGLQJUHJLRQV3ULPHUVZHUHGHVLJQHGIURPIODQN- OHIWYHQWULFXODUSRVWHULRUZDOOWKLFNQHVV• DQGIRXU ing intronic sequences for all exons of the Irx4 gene patients had apical hypertrophy. Basal left ventricular *HQ%DQNDFFHVVLRQQXPEHU$< *HQRPLF RXWIORZREVWUXFWLRQ JUDGLHQW•PP+J ZDVSUHV- '1$ IUDJPHQWV ZHUH DPSOLILHG E\ SRO\PHUDVH FKDLQ HQW LQ  SDWLHQWV   $OO FRQWURO VXEMHFWV KDG UHDFWLRQ 3&5 DQGVFUHHQHGE\VLQJOHVWUDQGFRQ- normal transthoracic echocardiograms. IRUPDWLRQSRO\PRUSKLVP 66&3 DQDO\VLV 7DEOH  Four polymorphisms were identified in the Irx4 $PSOLILHGIUDJPHQWVZHUHSXULILHGZLWKWKH4,$TXLFN JHQH QDPHO\ *!$ $!* *!$ DQG 3&5SXULILFDWLRQNLW 4LDJHQ*PE++LOGHQ*HUPDQ\  &!77KHORFDWLRQRIWKHVHVLQJOHQXFOHRWLGH 3&5IUDJPHQWVZLWKGLIIHUHQWLDOPLJUDWLRQZHUHFKDUDF- SRO\PRUSKLVPV 613V LQUHODWLRQWRJHQRPLFVWUXF- WHUL]HGE\'1$VHTXHQFLQJLQWKHSDWLHQWVDQGFRQWUROV ture of the Irx4 gene is shown in Fig. 1. In the public '1$VHTXHQFHVZHUHGHWHUPLQHGWKURXJKDQDXWRPDWHG GDWDEDVH GE613  QLQH SRO\PRUSKLVPV DUH SUHVHQW '1$VHTXHQFLQJV\VWHP *HQRVSKHUH%LRWHFKQRORJLHV in the protein coding region of the Irx4 gene, includ- 3DULV)UDQFH ,QDGGLWLRQWKH*!$DQG*!$ ing these four polymorphisms detected in our study: polymorphisms detected in Irx4 gene fragments were UV *!$ UV $!* UV FRQILUPHGE\GLJHVWLRQZLWK0VS$,DQG6DF,,UHVWULF- *!$  DQG UV &!7  7KH *WR$ tion enzymes, respectively. WUDQVLWLRQDWQXFOHRWLGH *!$ LQH[RQUHVXOW- Statistical analysis. Genotypic and allelic distri- ed in substitution of alanine for threonine at residue butions were compared using the chi-square test.  $OD7KU RIWKH,U[SURWHLQ7KHRWKHUSRO\- Continuous variables were compared with a two- morphisms did not cause residue change in the protein tailed t-test and expressed as means ± standard devia- 3UR3UR$OD$ODDQG*O\*O\  tion (SD), while categorical variables were compared Comparison of genotypic and allelic frequencies XVLQJWKHFKLVTXDUHWHVW$WZRWDLOHGp value of less of each polymorphism between patients and controls WKDQZDVFRQVLGHUHGVWDWLVWLFDOO\VLJQLILFDQW$OO VWDWLVWLFDO DQDO\VHV ZHUH SHUIRUPHG XVLQJ WKH 6366 LVVKRZQLQ7DEOH$OOHOLFGLVWULEXWLRQVGLGQRWGLIIHU software (Windows, version 10.0). significantly between patients and controls. &RPSDUHGWRSDWLHQWVZLWK*$DQG**JHQRW\SHV RESULTS WKH$$JHQRW\SHRI$!*SRO\PRUSKLVPZDVIRXQG The clinical, electrocardiographic, and echocardio- to be associated with a higher maximal left ventricle graphic characteristics of the patients with HC are outflow tract gradient (48±53 mmHg vs 24±35 mmHg; shown in Table 2. Sixty-four patients had asymmetric p=0.03), prolonged corrected QT dispersion (79±21 *HQHWLFDQDO\VLVRIWKH,U[JHQHLQK\SHUWURSKLFFDUGLRP\RSDWK\ 

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Figure 1.6FKHPDWLFVWUXFWXUHRIWKHKXPDQ,U[JHQHVKRZLQJWKHORFDOL]DWLRQRIIRXUSRO\PRUSKLVPVDQG DVVRFLDWHG'1$VHTXHQFLQJUHVXOWV3RO\PRUSKLVPVDUHFRXQWHGGRZQVWUHDPIURPWKH$7*WULSOHW $   FRGLQJIRUWKHVWDUWPHWKLRQLQH msec vs 63±18 msec; p=0.05), and albeit statistically Modifier genes are neither necessary nor sufficient insignificant, increased septal thickness (2.6±0.7 cm vs to cause HC, but they have a significant effect on the 2.2±0.5 cm; p=0.07). severity of the disease.[15] Recent reviews have pointed out that the influence of modifier genes and environ- DISCUSSION mental factors on the phenotypic expression of HC limit For the first time, we examined all exons in the human the utility of, and drawing conclusions from genotype- ,U[JHQHDQGLGHQWLILHGIRXUSRO\PRUSKLVPV *!$ phenotype correlation studies.[3,15] On the other hand, $!**!$DQG&!7 EXWZHFRXOGQRW VRPHPRGLILHUJHQHVVXFKDV$*SRO\PRUSKLVP find any mutations in the Irx4 gene associated with HC. LQWKHP\RVLQELQGLQJSURWHLQ& 0\%3& JHQHDQG

Table 3. Genotype and allele frequencies for polymorphisms of the Irx4 gene in patients and controls   3DWLHQWJURXS &RQWUROJURXS  *HQRW\SHV Q  Q  p *!$ **       $*       $$      *$DOOHOH    $!* $$      Q   $*       **      $*DOOHOH     *!$ **       *$      *$DOOHOH     &!7 &&       &7      &7DOOHOH    94 7UN.DUGL\RO'HUQ$Uü an insertion/deletion polymorphism in the angiotensin-1 of Cardiology/European Society of Cardiology clinical FRQYHUWLQJHQ]\PH $&( JHQHZHUHIRXQGWREHDVVR- expert consensus document on hypertrophic cardiomyo- ciated with the clinical phenotype of HC.[16-18] $OWKRXJK SDWK\$UHSRUWRIWKH$PHULFDQ&ROOHJHRI&DUGLRORJ\ RYHU  GLYHUVH PXWDWLRQV LQ 0\%3& DFFRXQW IRU Foundation Task Force on Clinical Expert Consensus approximately 20% to 25% of all HC cases,[3] it was Documents and the European Society of Cardiology &RPPLWWHH IRU 3UDFWLFH *XLGHOLQHV - $P &ROO &DUGLRO IRXQGWKDWWKH$*SRO\PRUSKLVPRI0\%3& 2003;42:1687-713. was also a genetic modifier for the development of left [16] 2. Maron BJ. Hypertrophic cardiomyopathy: a systematic ventricular wall thickness in patients with HC. UHYLHZ-$0$ In our study, no mutations were detected associat-  0DULDQ$-5REHUWV57KHPROHFXODUJHQHWLFEDVLVIRU HGZLWK+&EXWFRPSDUHGZLWKWKH*$DQG**JHQR- hypertrophic cardiomyopathy. J Mol Cell Cardiol 2001; W\SHVWKH$$JHQRW\SHRI$!*SRO\PRUSKLVP 33:655-70. was found to be associated with a higher maximal left 4. Bayrak F, Komurcu-Bayrak E, Mutlu B, Kahveci G, ventricle outflow tract gradient, a prolonged corrected %DVDUDQ<(UJLQHO8QDOWXQD19HQWULFXODUSUHH[FL- QT dispersion, and albeit statistically insignificant, tation and cardiac hypertrophy mimicking hypertro- phic cardiomyopathy in a Turkish family with a novel increased septal thickness in patients with HC. Thus, 35.$*PXWDWLRQ(XU-+HDUW)DLO WKH$!*SRO\PRUSKLVPRIWKH,U[JHQHPD\  %ODLU(5HGZRRG&$VKUDILDQ+2OLYHLUD0%UR[KROPH have a modifier effect on septal thickness resulting J, Kerr B, et al. Mutations in the gamma(2) subunit of in a prolonged corrected QT dispersion and higher $03DFWLYDWHGSURWHLQNLQDVHFDXVHIDPLOLDOK\SHUWUR- outflow gradients, but these data need to be validated phic cardiomyopathy: evidence for the central role of by further large scale studies. energy compromise in disease pathogenesis. Hum Mol In our study, mutation screening included the Genet 2001;10:1215-20. intron-exon boundaries and the coding regions of the  $UDG 0 %HQVRQ ': 3HUH]$WD\GH $5 0F.HQQD :-6SDUNV($.DQWHU5-HWDO&RQVWLWXWLYHO\DFWLYH Irx4 gene, but not other sites such as introns, and regu- $03NLQDVHPXWDWLRQVFDXVHJO\FRJHQVWRUDJHGLVHDVH ODWRU\HOHPHQWV3RVVLEOHSRO\PRUSKLVPVRUPXWDWLRQV mimicking hypertrophic cardiomyopathy. J Clin Invest of these sites might be the genetic modifier in the 2002;109:357-62. development of cardiac hypertrophy because animal 7. Danon MJ, Oh SJ, DiMauro S, Manaligod JR, Eastwood studies demonstrated that the absence of the Irx4 $1DLGX6HWDO/\VRVRPDOJO\FRJHQVWRUDJHGLVHDVH protein caused adult-onset cardiac hypertrophy.[9,11] with normal acid maltase. Neurology 1981;31:51-7. However, this needs to be substantiated by further  *HLHU & 3HUURW $ 2]FHOLN & %LQQHU 3 &RXQVHOO ' studies that include whole gene screening and expres- Hoffmann K, et al. Mutations in the human muscle sion pattern analysis. LIM protein gene in families with hypertrophic cardio- myopathy. Circulation 2003;107:1390-5. Our sample size may not be large enough to detect 9. Bao ZZ, Bruneau BG, Seidman JG, Seidman CE, Cepko mutations in the Irx4 gene; therefore, further studies CL. Regulation of chamber-specific gene expression in with broader patient populations are necessary. It may the developing heart by Irx4. Science 1999;283:1161-4. also be of value to analyze the Irx4 gene in patients 10. Bruneau BG, Bao ZZ, Tanaka M, Schott JJ, Izumo S, without sarcomere gene mutations. Cepko CL, et al. Cardiac expression of the ventricle- This is the first human study investigating the specific homeobox gene Irx4 is modulated by Nkx2-5 and dHand. Dev Biol 2000;217:266-77. association between possible mutations in the Irx4 11. Bruneau BG, Bao ZZ, Fatkin D, Xavier- gene and HC. Four polymorphisms were identified Neto J, Georgakopoulos D, Maguire CT, et al. ZLWKLQWKH,U[JHQH7KH$$JHQRW\SHRI$!* Cardiomyopathy in Irx4-deficient mice is preceded polymorphism was found to be associated with a by abnormal ventricular gene expression. Mol Cell higher maximal left ventricle outflow tract gradient Biol 2001;21:1730-6. and prolonged corrected QT dispersion. &KULVWRIIHOV 90 .HLMVHU $* +RXZHOLQJ $& &ORXW Acknowledgments '( 0RRUPDQ $) 3DWWHUQLQJ WKH HPEU\RQLF KHDUW identification of five mouse Iroquois homeobox genes This work was supported by the Research Fund of in the developing heart. Dev Biol 2000;224:263-74. ,VWDQEXO8QLYHUVLW\3URMHFWQXPEHU +RXZHOLQJ$&'LOGURS53HWHUV70XPPHQKRII- 0RRUPDQ$)5XWKHU8HWDO*HQHDQGFOXVWHUVSHFLILF REFERENCES expression of the Iroquois family members during 1. Maron BJ, McKenna WJ, Danielson GK, Kappenberger mouse development. Mech Dev 2001;107:169-74. /-.XKQ+-6HLGPDQ&(HWDO$PHULFDQ&ROOHJH .OXHV +* 6FKLIIHUV $ 0DURQ %- 3KHQRW\SLF VSHF- *HQHWLFDQDO\VLVRIWKH,U[JHQHLQK\SHUWURSKLFFDUGLRP\RSDWK\ 

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