we investigated four single nucleotide polymorphisms in the gene - encoding angiotensin converting enzyme 2 ( ace2 ) , a gene on the x - chromosome whose raas product modulates cardiac structure and function , for association with hypertrophic traits in 227 individuals belonging to 22 hcm families with known founder disease - causing mutations .
after adjustment for hypertrophy - influencing factors , including the particular disease - causing mutation , we find association between the g - allele of rs879922 and increased left ventricular mass ( effect size : 18.7  g ) , maximum interventricular septal thickness ( effect size : 1.9  mm ) as well as maximum posterior wall thickness ( effect size : 0.7  mm ) .
these data demonstrate that ace2 plays a role in modifying the extent of hypertrophy that develops in hcm , independent of the effect of the disease - causing mutation or blood pressure .
hypertrophic cardiomyopathy ( hcm ; omim # 192600 ) is a primary cardiac muscle disorder classically caused by mutations ( > 400 ) in genes encoding components of the thick and thin filaments of the cardiac sarcomere ( http://genetics.med.harvard.edu/~seidman/cg3/index.html ) .
extreme variability of the hypertrophic phenotype is a hallmark feature of hcm and has been attributed in part to allelic effects of the disease - causing gene .
evidence from human and transgenic animal studies indicates that the degree of hypertrophy that develops in response to the causal mutation is modified by environmental factors ( including demographic factors such as age , sex and body size ) , as well as by other genetic factors ( semsarian et al .
genes within the renin - angiotensin - aldosterone system ( raas ) have been investigated as modifiers of the hypertrophic variability in hcm in recent years ( marian 2002 ; ortlepp et al . 2002 ) , and much emphasis has been placed on the possible role of the angiotensin - converting - enzyme ( ace ) i in the development of hypertrophy .
however , subsequently , a second ace , aceii , has been discovered and its product , viz .
angiotensin-(1 - 7 ) , has been found to antagonize the vasoconstriction mediated by its substrate , angiotensin ii .
lieb et al . found that minor alleles of four single nucleotide polymorphisms ( snps ) in the x - chromosomally located aceii gene ( ace2 ) were associated with increases in hypertrophic parameters , viz .
left ventricular mass indexed to body size ( lvmi ) , as well as end - diastolic interventricular septal wall thickness ( ivs ) in males , but not in females ( lieb et al .
. also reported that minor alleles of other snps in ace2 were again associated with increased ivs in male but not female hcm patients ( wang et al .
, it is possible that the effect of the unknown hcm - causing mutations in the case cohort could confound the results .
hence , we aimed to investigate the role of ace2 variants in the extent of hypertrophy that develops in genotype - known hcm patients belonging to families in which any of three south african hcm founder mutations segregate .
the study was approved by the university of stellenbosch health sciences faculty s institutional review board ( n04/03/062 ) .
twenty - two consecutively referred index cases bearing one of the previously reported south african hcm - founder mutations ( 7 r92wtnnt2 , 3 r403wmyh7 and 11 a797tmyh7 ) were identified and pedigree tracing was performed ( revera et al .
individuals who gave informed written consent were tested for carrier - status for each of the three founder mutations , as previously described ( moolman - smook et al .
blood pressure was taken , twice : in the sitting position , after 5  min of bed rest , and the second measurement was used .
individuals were coded as hypertensive if they had systolic blood pressure  140  mm  hg , or diastolic blood pressure  90 , or were on anti - hypertensive medication .
medical history , and demographic data ( age , sex , height , weight ) were recorded for each participant .
we performed standard electrocardiography on a mac1200st after 5  min of rest in the supine position , and from this derived resting heart rate .
echocardiography was performed anew on all participating individuals , using a standardized procedure to measure the wall thickness in 16 left ventricular wall segments , by a single experienced echocardiographer ( mr ) who was blinded to mutation status , according to recommendations of the american society of echocardiography ( schiller et al .
lvm was calculated using the american society of echocardiography s recommended formula for estimation of lvm from 2d - lv linear dimensions:\documentclass[12pt]{minimal } 
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 \begin{document}$$ { \text{lvm } } = 0.8\ , \times \left ( { 1.04\left [ { \left ( { { \text{lvidd } } + { \text{pwtd } } + { \text{swtd } } } \right)^{{3 } } -\left ( { { \text{lvidd } } } \right)^{{3 } } } \right ] } \right ) + 0.6\,{\text{g}}. $ $ \end{document } as the ld structure of this ( and other ) regions of the genome has not been reported in the study population , we relied on density selection of snps .
rs1978124 ( intron 1 ) , rs2285666 ( intron 3 ) , rs879922 ( intron 11 ) and rs4646179 ( exon 16 ) , spread through the length of ace2 at an average spacing of ~8  kb , were selected from dbsnp .
these snps were chosen because they had been validated by frequency , had been found associated with cardiovascular traits in previous studies ( lieb et al .
ace2 genomic regions containing the variants of interest were pcr amplified from subjects genomic dna using standard methodology ( moolman - smook et al . 1999 ) ; primers and annealing temperature used are given in table  1 .
variants were genotyped by allele - specific restriction enzyme digestion of the pcr amplicons and fragment sizes detected by gel electrophoresis ( table  1 ) .
ten percent of samples , randomly selected for each amplicon , were nucleotide sequenced to verify genotyping results . furthermore ,
mendelian inheritance within families was assessed by pedstats , using x - chromosome settings , and inconsistencies were resolved by re-genotyping.table  1summary of pcr - based allele - specific restriction enzyme analysis used for genotyping of ace2 variantsvariantprimers ( 53)restriction enzymeamplicon size ( bp)major allele sizes ( bp)minor allele sizes ( bp)detection methodrs1978124f : tcttcctggctccttctcagcsau96i1,044 t ( 1044)c ( 962 , 82)1.5% agarose electrophoresisr : accacaatggcagagaaagggrs2285666f : gtttgtaacccagataatccalui130c ( 130)t ( 78 , 52)12% pager : gttgaaacacacatatctgcrs879922f : ttgtgttaagatcttgtcccbfai188c ( 188)g ( 146 , 42)12% pager : aataaactgagctccagcrs4646179f : tacagggaggaggatgtgcghpych4iv680 t ( 328 , 229 , 123)c ( 328 , 152 , 123 , 77)2.5% agarose electrophoresisr : tgatggcaatacctgtccacgbp base pairs , page polyacrylamide gel electrophoresis summary of pcr - based allele - specific restriction enzyme analysis used for genotyping of ace2 variants bp base pairs , page polyacrylamide gel electrophoresis quantile normalization was used to convert each trait to approximate normality ( pilia et al .
2006 ) . associations of ace2 variants with heritable hypertrophy traits , viz . left ventricular mass ( lvm ) , maximum interventricular septal thickness ( maxivs ) and maximum posterior wall thickness ( maxpw ) , were assessed with specialized mixed - effects models utilizing per - individual random effects which are correlated according to kinship coefficients ( r package kinship , function lmekin ) .
furthermore , concomitant adjustment was made for factors known to influence cardiac hypertrophy , viz .
age , sex , body surface area , heart rate , systolic and diastolic blood pressure , hypertension diagnosis , as well as the identity of the disease - causing mutation , by including these as covariates in the analyses .
we then formally tested the interaction between sex and genotype , after modeling the genotypes as the number of minor alleles ( additive term ) , and a dominance term , which was only non - zero for heterozygous females ( cordell and clayton 2005 ) .
this model is equivalent to modeling the genotypes as categories , labeling the male genotypes differently to the female genotypes .
when the interaction between sex and genotype was not statistically significant , it was discarded from the model . the dominance term was similarly discarded if not significantly different from zero .
as effect sizes derived from analysis of transformed data can not be interpreted in terms of the original measurement units , we also modeled the raw data in terms of the genotypes of the associated variant rs879922 , with adjustment for confounders and to obtain informative effect sizes . because there were only two gg homozygotes in the group , we combined them with the heterozygotes to estimate the effect of at least one g - allele .
linkage disequilibrium between the four markers and hardy  weinberg equilibrium in the study cohort was assessed by haploview vs3.32 .
the study was approved by the university of stellenbosch health sciences faculty s institutional review board ( n04/03/062 ) .
twenty - two consecutively referred index cases bearing one of the previously reported south african hcm - founder mutations ( 7 r92wtnnt2 , 3 r403wmyh7 and 11 a797tmyh7 ) were identified and pedigree tracing was performed ( revera et al .
individuals who gave informed written consent were tested for carrier - status for each of the three founder mutations , as previously described ( moolman - smook et al .
blood pressure was taken , twice : in the sitting position , after 5  min of bed rest , and the second measurement was used .
individuals were coded as hypertensive if they had systolic blood pressure  140  mm  hg , or diastolic blood pressure  90 , or were on anti - hypertensive medication .
medical history , and demographic data ( age , sex , height , weight ) were recorded for each participant .
we performed standard electrocardiography on a mac1200st after 5  min of rest in the supine position , and from this derived resting heart rate .
echocardiography was performed anew on all participating individuals , using a standardized procedure to measure the wall thickness in 16 left ventricular wall segments , by a single experienced echocardiographer ( mr ) who was blinded to mutation status , according to recommendations of the american society of echocardiography ( schiller et al .
lvm was calculated using the american society of echocardiography s recommended formula for estimation of lvm from 2d - lv linear dimensions:\documentclass[12pt]{minimal } 
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 \begin{document}$$ { \text{lvm } } = 0.8\ , \times \left ( { 1.04\left [ { \left ( { { \text{lvidd } } + { \text{pwtd } } + { \text{swtd } } } \right)^{{3 } } -\left ( { { \text{lvidd } } } \right)^{{3 } } } \right ] } \right ) + 0.6\,{\text{g}}. $ $ \end{document }
as the ld structure of this ( and other ) regions of the genome has not been reported in the study population , we relied on density selection of snps .
rs1978124 ( intron 1 ) , rs2285666 ( intron 3 ) , rs879922 ( intron 11 ) and rs4646179 ( exon 16 ) , spread through the length of ace2 at an average spacing of ~8  kb , were selected from dbsnp . these snps were chosen because they had been validated by frequency , had been found associated with cardiovascular traits in previous studies ( lieb et al .
ace2 genomic regions containing the variants of interest were pcr amplified from subjects genomic dna using standard methodology ( moolman - smook et al .
variants were genotyped by allele - specific restriction enzyme digestion of the pcr amplicons and fragment sizes detected by gel electrophoresis ( table  1 ) .
two independent observers , blinded to phenotypic data , performed allele - calling . ten percent of samples , randomly selected for each amplicon , were nucleotide sequenced to verify genotyping results . furthermore ,
mendelian inheritance within families was assessed by pedstats , using x - chromosome settings , and inconsistencies were resolved by re-genotyping.table  1summary of pcr - based allele - specific restriction enzyme analysis used for genotyping of ace2 variantsvariantprimers ( 53)restriction enzymeamplicon size ( bp)major allele sizes ( bp)minor allele sizes ( bp)detection methodrs1978124f : tcttcctggctccttctcagcsau96i1,044 t ( 1044)c ( 962 , 82)1.5% agarose electrophoresisr : accacaatggcagagaaagggrs2285666f : gtttgtaacccagataatccalui130c ( 130)t ( 78 , 52)12% pager : gttgaaacacacatatctgcrs879922f : ttgtgttaagatcttgtcccbfai188c ( 188)g ( 146 , 42)12% pager : aataaactgagctccagcrs4646179f : tacagggaggaggatgtgcghpych4iv680 t ( 328 , 229 , 123)c ( 328 , 152 , 123 , 77)2.5% agarose electrophoresisr : tgatggcaatacctgtccacgbp base pairs , page polyacrylamide gel electrophoresis summary of pcr - based allele - specific restriction enzyme analysis used for genotyping of ace2 variants bp base pairs , page polyacrylamide gel electrophoresis
quantile normalization was used to convert each trait to approximate normality ( pilia et al .
2006 ) . associations of ace2 variants with heritable hypertrophy traits , viz . left ventricular mass ( lvm ) , maximum interventricular septal thickness ( maxivs ) and maximum posterior wall thickness ( maxpw ) , were assessed with specialized mixed - effects models utilizing per - individual random effects which are correlated according to kinship coefficients ( r package kinship , function lmekin ) .
furthermore , concomitant adjustment was made for factors known to influence cardiac hypertrophy , viz .
age , sex , body surface area , heart rate , systolic and diastolic blood pressure , hypertension diagnosis , as well as the identity of the disease - causing mutation , by including these as covariates in the analyses .
we then formally tested the interaction between sex and genotype , after modeling the genotypes as the number of minor alleles ( additive term ) , and a dominance term , which was only non - zero for heterozygous females ( cordell and clayton 2005 ) .
this model is equivalent to modeling the genotypes as categories , labeling the male genotypes differently to the female genotypes .
when the interaction between sex and genotype was not statistically significant , it was discarded from the model . the dominance term was similarly discarded if not significantly different from zero .
as effect sizes derived from analysis of transformed data can not be interpreted in terms of the original measurement units , we also modeled the raw data in terms of the genotypes of the associated variant rs879922 , with adjustment for confounders and to obtain informative effect sizes . because there were only two gg homozygotes in the group , we combined them with the heterozygotes to estimate the effect of at least one g - allele .
linkage disequilibrium between the four markers and hardy  weinberg equilibrium in the study cohort was assessed by haploview vs3.32 .
three hundred and twenty individuals belonging to 7 r92wtnnt2 , 3 r403wmyh7 and 12 a797tmyh7 families were genotyped for variants in ace2 ; of these , 227 individuals , including all hcm mutation - carriers , consented to clinical investigation ; clinical and demographic traits of these individuals are summarized in table  2.table  2basic statistics of relevant clinical and demographic traits in the study cohort , stratified by hcm - mutation statushcm - mutation bearerscontrolsn  =  127n  =  100median(q1,q3)median(q1,q3)age ( years)41(25,53)41(29,51)bsa ( kg / m)1.8(1.6,2.0)1.8(1.7,2.0)systbp ( mm  hg)120(110,130)120(110,130)diastbp ( mm  hg)80(70,80)80(70,90)heart rate ( bpm)66(60,75)69(62,76)lvm ( g)166(125,218)126(103,158)mivst ( mm)13.9(10.4,19.6)10.2(9.0,11.3)mpw ( mm)10.1(9.0,11.6)9.0(8.0,9.7)because of the skewness of some distributions , data are summarised here as median ( interquartile range ) , and were quantile normalized prior to association analysis(q1 , q3 )  =  q1 , first quartile , q3 , third quartile ; bsa , body surface area ; syst , systolic ; bp , blood pressure ; diast , diastolic ; bpm , beats per minute ; lvm , left ventricular mass ; g , grams ; mivst , maximum interventricular septal thickness ; mm , millimeter ; mpw , maximum posterior wall thickness basic statistics of relevant clinical and demographic traits in the study cohort , stratified by hcm - mutation status because of the skewness of some distributions , data are summarised here as median ( interquartile range ) , and were quantile normalized prior to association analysis ( q1 , q3 )  =  q1 , first quartile , q3 , third quartile ; bsa , body surface area ; syst , systolic ; bp , blood pressure ; diast , diastolic ; bpm , beats per minute ; lvm , left ventricular mass ; g , grams ; mivst , maximum interventricular septal thickness ; mm , millimeter ; mpw , maximum posterior wall thickness all markers were in hardy  weinberg equilibrium in the unrelated individuals and there was no relevant correlation between the snps ( all r    0.1 ) .
genotype frequencies of the markers in the study population are given in table  3.table  3genotype frequencies for ace2 variants in the complete study cohort , stratified by gendermalefemalecgcccgggrs19781240.380.620.160.310.53rs22856660.810.190.590.380.03rs8799220.770.230.500.490.01rs46461790.980.020.980.020.00 genotype frequencies for ace2 variants in the complete study cohort , stratified by gender statististically , there were no significant interactions between sex and genotype , so that no sex - specific effects of alleles were observed ( results not shown ) . as the dominance term was not significantly different from zero ,
we could model the genotypes as single numerical factors , specified as the number of minor - alleles carried by the individual ; thus males had a maximum of one of these alleles and females a maximum of two (  additive  coding ) ( cordell and clayton 2005 ) .
the p values for significance tests of the additive term , after removing the dominance term from the model , are shown in table  4 .
while there was no evidence for association between hypertrophy traits and ace2 snps rs1978124 , rs2285666 or rs4646179 , the g - allele of rs879922 was found to have a significant effect on all three of the investigated hypertrophic traits , increasing each by a modest amount .
the effect sizes of the g - allele on untransformed , covariate - adjusted traits were 18.7  g for lvm , 1.9  mm for maxivs and 0.7  mm for maxpw ( table  4).table  4effect size ( beta ) , standard error ( se ) and p value for tests of association of variants in ace2 snplvmechomax ivstmax pwbetasep valuebetasep valuebetasep valuers19781248.05.90.11901.140.620.24480.3150.2340.0772rs22856666.78.50.65440.020.800.77960.2460.2760.1825rs87992218.79.40.02051.870.870.03930.6570.3050.0090rs464617926.927.00.48133.712.560.29990.1540.8930.6102effect size and standard error are given in original units of measurement for traits , for ease of interpretation , while p values are for tests on quantile normalised traits .
beta is the effect on the trait of adding a g - allelelvm left ventricular mass , maxivst maximum interventricular septal thickness , maxpw maximum posterior wall thickness effect size ( beta ) , standard error ( se ) and p value for tests of association of variants in ace2   effect size and standard error are given in original units of measurement for traits , for ease of interpretation , while p values are for tests on quantile normalised traits .
beta is the effect on the trait of adding a g - allele lvm left ventricular mass , maxivst maximum interventricular septal thickness , maxpw maximum posterior wall thickness
in this study , we used hcm families , in which the disease is caused by defined founder mutations , to assess the role of variants in ace2 in hypertrophy development , independent of factors that are known to influence this phenomenon .
we find that the g - allele of rs879922 is associated with increased hypertrophy , viz .
lieb et al . previously reported association of the rs879922 g - allele , singly as well as within a four - marker haplotype ( with rs4646156 , rs4240157 and rs233575 ) , with modestly increased lvm and maxivs in the general male population in augsburg , germany ( lieb et al .
wang et al . recently reported association of other ace2 variants , rs2106809 and rs663677 , with maxivs , again only in a group of male hcm patients , with unknown primary disease - causing mutations ( wang et al .
although ace2 maps to a hypertension quantitative trait locus identified in three different rat models of hypertension , no association between ace2 variants and hypertension has been found to date ( benjafield et al .
; wang et al . 2008 ) . in our study , association with hypertrophy was also independent of blood pressure per se or diagnosis of hypertension . taken together , our data indicate a role for variation in ace2 in modifying the development of left ventricular hypertrophy .
furthermore , the results suggest that the genetic modifiers of the cardiac hypertrophy that develops in response to a primary hcm - causing mutation may also be involved in hypertrophy development in the general population . the effect size of the g - allele of rs879922 is modest , as has been previously described for other raas gene variants ( mayosi et al .
2003 ) , which may suggest that the hypertrophic phenotype is the cumulative result of a number of genes of moderate effect . as rs879922 occurs within an intron and
does not have any obvious functional role , it is possible that the association noted in this study , as well as that of lieb et al .
the extent of variation in this gene and the ld distribution across the genome in the study population is unknown .
however , rs879922 tags at least 28 additional hapmap snps in the ceu , yri and jpt  +  chb populations . in the ceu and yri hapmap populations , variation in ace2 is predominantly captured by two and three haplotype blocks , respectively ; rs879922 is located within a haplotype block involving the 3 approximately third of the gene , which may indicate that the functional variant lies in this region of the gene . due to the highly variable distribution of
left ventricular hypertrophy in hcm , it is difficult to distinguish a single echocardiographic measure that accurately quantifies the extent of hypertrophy in all patients . although lvm is most often used to quantify hypertrophy in hcm , it is known to be an inaccurate measure when echocardiographically derived , due to the asymmetric nature of hypertrophy in hcm .
we did not employ bonferroni correction for multiple testing , as the traits investigated are highly correlated , and such correction is considered over - conservative , risking the rejection of important findings , while bayesian methods for correction rely on knowledge of prior probability of involvement , which is currently unknown for most genetic variants ( campbell and rudan 2002 ) .
however , even if the over - stringent bonferroni correction for four independent tests ( for the four markers ) were performed , the association noted for rs879922 with maxpw would remain significant , and that with lvm remain a trend .
although the number of subjects assessed in this study is modest , our data adds to previous investigations in confirming the involvement of ace2 in the development of cardiac hypertrophy , independent of blood pressure or indeed other hypertrophy covariates , in hcm patients .
furthermore , in our study , the use of data derived from hcm families carrying known , founder mutations afforded benefits over case - control studies , such as additional control of genotyping accuracy , control over the variability introduced by distinct primary disease - causing mutations , and avoidance of the confounding effect of occult population stratification .
furthermore , our study combined with that of lieb et al . and wang et al . supports a more consistent role for the recently identified component of raas , aceii , than the discordant findings for the original acei , in modulating cardiac hypertrophy , and in particular in modifying the extent of hypertrophy that develops in response to hcm - causing mutations .
 hypertrophic cardiomyopathy , a common , inherited cardiac muscle disease , is primarily caused by mutations in sarcomeric protein - encoding genes and is characterized by overgrowth of ventricular muscle that is highly variable in extent and location . 
 this variability has been partially attributed to locus and allelic heterogeneity of the disease - causing gene , but other factors , including unknown genetic factors , also modulate the extent of hypertrophy that develops in response to the defective sarcomeric functioning . 
 components of the renin - angiotensin - aldosterone system are plausible candidate hypertrophy modifiers because of their role in controlling blood pressure and biological effects on cardiomyocyte hypertrophy . 
Introduction
Methods
Subjects
Clinical evaluation
None
Statistical analyses
Results
Discussion