© 1997 American Heart Association, Inc.
Articles |
Genetic Contribution of the Endothelial Constitutive Nitric Oxide Synthase Gene to Plasma Nitric Oxide Levels
From the Department of Cardiovascular Medicine, Prince Henry/Prince of Wales Hospitals (X.L.W., A.S.S., Jun W., R.B.B., D.E.L.W.), and the Department of Medicine, St George Hospital, University of New South Wales (M.C.M., J.B., L.A.), Sydney, Australia; and the Department of Genetics, Southwest Foundation of Biomedical Research, San Antonio, Texas (Jian W.).
Correspondence to Dr X.L. Wang, Department of Cardiovascular Medicine, Clinical Sciences Building, Prince Henry Hospital, Little Bay, NSW 2036, Australia. E-mail x.l.wang{at}unsw.edu.au
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Abstract Nitric oxide (NO) has an important physiological role in regulating vascular tone and is also relevant to many pathological processes including hypertension and atherosclerosis. Endothelial constitutive nitric oxide synthase (ecNOS) is the key enzyme in determining basal vascular wall NO production. We used a combination of maximum-likelihood-based statistical genetic methods to explore the contributions of the ecNOS gene and other unmeasured genes to basal NO production measured by its metabolites (NOx: nitrite and nitrate) in 428 members of 108 nuclear families. Our initial quantitative genetic analysis estimated that approximately 30% of the variance in fasting NOx levels is due to genes (
2[1]=16.04,
P=.000062). Complex segregation analysis detected
the effects of both a single locus and residual polygenes on
NOx levels, and measured genotype analysis
showed that plasma NOx levels in those homozygous for the
rare allele (64.9±7.8 µmol/L) were significantly higher
(P=.000242) than those homozygous for the common allele
(30.2±3.1 µmol/L). The results of the variance component
linkage analysis were consistent with linkage of a
quantitative trait locus in or near the ecNOS gene to variation in
plasma NOx levels (P=.0066). While many
environmental factors have been shown to alter transiently plasma
NOx levels, our study is the first to identify a
substantial effect of the ecNOS locus on the variance of plasma
NOx, ie basal NO production. This finding may be
relevant to atherogenesis and NO-related disorders.
Key Words: nitric oxide • endothelial constitutive nitric oxide synthase • DNA polymorphism • quantitative linkage analysis • major locus effect
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Introduction |
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Nitric oxide is biosynthesized from L-arginine by a family of NOSs in many tissues.1 Once generated, NO can interact with a number of molecular targets, and these determine the profile of NO as a major biological mediator, modulator, and effector.2 Three NOSs are responsible for NO biosynthesis in various tissues.1 Neural constitutive NOS (ncNOS, or NOS1) is expressed in neurons and is responsible for the physiological production of NO in neural tissue. The gene is located on chromosome 12q24.2. ecNOS, or NOS3, is mainly confined to endothelium and produces NO, which contributes to the regulation of vascular tone. The gene is located at 7q35-36. The NO produced by these two constitutive NOSs participates in normal physiology. However, inducible NOS (iNOS, or NOS2, 17 cen-q12) is not normally expressed in most cells but may be induced in pathological processes as seen, for example, during inflammation.1
Circulating NO is mainly produced by ecNOS and has an important role in regulating blood flow, particularly coronary flow.3 4 5 6 Reduction in basal NO release may predispose to hypertension, thrombosis, vasospasm, and atherosclerosis.3 4 5 6 7 8 9 In animal models, restoration of NO activity can induce regression of preexisting intimal lesions.10 On the other hand, high circulating NO levels, which occur with excess iNOS expression under pathological conditions, are generally toxic.1 2 There are data indicating that markedly elevated NO levels are associated with endotoxic shock and exaggerated inflammation reactions11 and may lead to acute hepatic dysfunction,12 as well as contribute to the pathogenesis of glomerulonephritis13 and predispose to asthma,14 cardiomyopathy,15 and a number of other disorders.3 The measurement of NO itself is difficult because of its very short half-life, and the stable metabolites of NO (NOx), have been frequently used as a reliable plasma measurement of NO production.16 17 18 19 20
The factors influencing an individual's continuous basal NO production are not clear.1 2 21 More specifically, the association between basal circulating NO levels and genotypic or phenotypic in vivo variations in ecNOS, the key regulator of the basal NO production in vasculature, is not understood. Determinations of in vivo human ecNOS enzymatic activity and its association with plasma NO are difficult because vascular tissue homogenates would be required. As ecNOS genomic DNA can be readily obtained and analyzed from peripheral nucleated blood cells, it is possible to assess the association between plasma NO and ecNOS at the DNA level. The definition of a genetic contribution to basal NO production is important for studies of the potential roles of NO and ecNOS in atherogenesis and hypertension, in both of which there is clustering in families.
Recently, we have reported an association between the ecNOS gene and an increased risk of coronary artery disease using a DNA polymorphism at intron 4 of the gene as a molecular marker.22 In the present study, we used this polymorphic marker to test the specific hypothesis that variation at the ecNOS locus contributes to quantitative phenotypic variation in circulating plasma NO levels, as estimated by NOx levels. Our results indicate that there is a significant genetic contribution to plasma NOx levels.
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Methods |
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Nuclear Families in the Study
We collected blood samples from 428 members of 108 nuclear families. These families were healthy volunteers recruited from our ongoing Heart Health Education Program for family-based primary coronary prevention. They were selected for the current study on the basis that all were white of European origin, and residing in Sydney; none were current smokers. They were advised to remain on their usual diet before the blood collection and all were healthy at the time of study. Written consent was obtained from every parent. The study was approved by the Ethics Committee of the University of New South Wales.
A 4-mL venous blood sample was drawn into an EDTA sample tube after an overnight fast (12 to 14 hours). The blood sample was centrifuged within 2 hours and plasma stored at -70°C in aliquots until analysis. DNA was extracted from the frozen cellular blood component by a salting-out method.22 The extracted DNA was stored at 4°C until analysis.
Genotyping of the 27-bp Repeat Polymorphism in Intron 4 of the
ecNOS Gene
A polymerase chain reaction method was used for the genotyping
of the repeat polymorphism as described previously.22
We used oligonucleotide primers that flank the region
of the 27-bp direct repeat in intron 4 of the ecNOS gene. The PCR
products were electrophoresed on 8% polyacrylamide gels
and visualized by silver staining. There are two alleles differing
by one repeat, ie, 27 bp in size. We have denoted these two alleles
as ecNOS4a for four repeats and ecNOS4b for five repeats; ecNOS4b is
the common allele.22
Determination of Plasma NOx Levels
Since NO is unstable and quickly oxidized to nitrate and nitrite
after production, to estimate plasma NO levels, circulating
NOx levels were determined using a modified method
described by Moshage et al.16 Nitrate was measured as
nitrite after enzymatic conversion by nitrate reductase and nitrite was
measured after deproteinization using the Griess color
reaction,18 which was read at a wavelength of 540 nm.
Values obtained by this procedure represent the sum of nitrite
and nitrate derived from NO. The detection limit of the assay and the
recovery rates for nitrate and nitrite were similar to those described
by Moshage et al.16 The precision profile of the assay was
assessed by the intra-assay and interassay coefficients of variation,
and they were 2.1% and 4.3%, respectively, in our laboratory.
Statistical Genetic Analysis
There were two principal objectives of the statistical genetic
analysis of circulating NOx levels in these 108
human nuclear families. The first objective included the detection and
characterization of genetic contributions to the phenotypic variance in
circulating levels of NOx. To achieve this objective, we
employed quantitative genetic analysis and complex segregation
analysis. The second objective was to test the specific
hypothesis that variation at the ecNOS4 locus contributes to phenotypic
variation in circulating NOx levels. To achieve this
objective, we employed a variance component linkage analysis
and measured genotype analysis (see "Appendix" for
details of the analysis).
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Results |
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There were 428 members of 108 nuclear families included in the current study. The average number of children was 2.3±1.5 with a range of 1 to 4 in each family. The ages (mean±SD) for fathers, mothers, boys, and girls were 43.5±6.1, 39.8±4.6, 11.1±3.2, and 11.0±3.2 years, respectively. Creatinine levels were within the normal range for all subjects in the study. The mean±SD fasting plasma NOx levels were not different between fathers, mothers, boys, and girls and were, respectively, 36.9±17.8, 33.6±19.4, 33.4±17.0, and 29.6±14.2 µmol/L.
Quantitative Genetic Analysis
The maximum-likelihood parameter estimates and their
SEs for the more general additive genetic (often referred to as
"polygenic") model and the restricted sporadic model are
presented in Table 1
. In the
former, the phenotypic variance is attributable to the effects of
additive genes, selected covariates, and random environmental factors;
in the latter, it is attributable to the covariates and random
environmental factors only. The results of the likelihood ratio tests
comparing these two models indicate that a significant proportion of
the residual phenotypic variance in plasma NOx levels,
approximately 30%, is due to the additive effects of genes
(2[1]=16.04, P=.000062).
Inclusion of sex and the age-by-sex terms as covariates does not
contribute significantly to the likelihood of this polygenic model
(P>.10).
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Complex Segregation Analysis
Likelihood ratio test screens of the five potential covariates
detected significant effects on variation in NOx levels for
creatinine and apolipoprotein A-I levels only. Inclusion of
both these covariates in the maximum-likelihood models reduced the
sample size from 428 individuals in 108 pedigrees to 291 individuals in
88 pedigrees.
The results of the complex segregation analysis,
summarized in Table 2, are
consistent with the detection of the effect of a single locus
on quantitative variation in plasma NOx levels in this
population. With the exception of the genetic (Mendelian) mixture
model, the ln likelihoods of all restricted alternate models tested
were significantly worse than that of the unrestricted general model.
Given their SEs, the maximum-likelihood estimates of the transmission
probabilities in the general model (
AA=0.95±0.10,
Aa=0.52±0.11, and aa,=0.24±0.27)
correspond to those expected under Mendelian segregation (ie, 1.0, 0.5,
and 0). The best-fitting model was a codominant mixture model. The
common allele segregating at this locus (designated A in
this analysis) produces lower NOx levels and has a
relative frequency of pA=0.64. Given the estimated
genotypic means, this allele appears to be dominant to the higher
NOx allele (a) in this model. Assuming
Hardy-Weinberg equilibrium, the approximate expected genotypic
proportions are 0.41 (AA), 0.46 (Aa),
0.13(aa).
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Measured Genotype Analysis
Table 3 shows the maximum-likelihood
estimates of the parameters of the measured
genotype model. The effects of sex and the age-by-sex terms on
the likelihood of the measured genotype model were not
significant (P>.10). Likelihood ratio tests reveal a
significant measured gene effect on plasma NOx levels
(2[3]=19.25, P=.000242).
The pattern of genotypic means of NOx levels is
consistent with the ecNOS4a/a homozygote exhibiting
significantly higher levels of circulating plasma NOx than
the other two genotypes, which have similar values (Table 3 and
the Figure). In this sample of nuclear
families, the measured gene accounts for 25% of the total phenotypic
variance in plasma NOx levels; residual additive genes and
environmental factors account for approximately 7% and 68%,
respectively.
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Variance Component Linkage Analysis
The results of the variance components genetic analysis of
plasma NOx levels are summarized in Table 4. Sex, age-by-sex, and
age2-by-sex exerted no significant effects on the
likelihoods of either the restricted or general models and consequently
were removed prior to a second maximization series, and this is
summarized in the table. Maximization of the restricted model on the
nuclear family data clearly identifies a significant heritable
component to quantitative variation in plasma NOx levels.
The proportion of the phenotypic variance attributable to the additive
effects of genes is 31.5% (
=16.338, P=.000053). As
disclosed by the likelihood ratio test comparing the general model and
restricted models, a significant proportion of the phenotypic variance,
25.3%, is attributable to variation at the ecNOS4 locus. Approximately
10% and 64% of the phenotypic variance is attributable, respectively,
to residual additive genetic effects and to random environmental
effects.
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Discussion |
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Our study is the first to detect the effect of genes on quantitative variation in plasma NOx levels in healthy human subjects who were randomly ascertained with respect to the NOx phenotype and to identify a specific quantitative trait locus responsible for a significant portion of that effect. The results of our quantitative genetic analysis indicate that a moderate but significant proportion of the phenotypic variance in circulating plasma NOx levels in this population is attributable to the additive effects of genes. This conclusion is supported and expanded by the results of our subsequent complex segregation analysis, conducted on a subsample of the subject population, that also detects these additive genetic effects plus the influence of a single locus, with apparent dominance effects, on variation in plasma NOx levels.
The measured genotype analysis provides initial evidence for the involvement of the ecNOS locus in the regulation of plasma levels of NOx in these families. The location in a noncoding region of the candidate gene minimizes the likelihood of a functional association between the polymorphic marker itself and plasma NOx levels in this study. The 25% contribution of this marker to the phenotypic variance in the study, estimated in the measured genotype analysis, represents a minimum estimate of the variance in plasma NOx levels attributable to the associated quantitative trait locus.
The results of the variance component linkage analysis indicate that the marker is most likely physically linked to the quantitative trait locus. They also indicate that the marker is most likely physically linked to the quantitative trait locus and provide a more valid estimate of the proportion of the variance attributable to the quantitative trait locus in these families. Our results indicate that the quantitative trait locus is linked to the ecNOS4 polymorphism. Given that many environmental factors have been shown to alter transiently plasma NOx levels,1 2 3 the effect of the detected locus, accounting for over 25% of the phenotypic variation in this trait, is substantial and is likely to have relevance to the pathogenesis of NO related disorders such as atherosclerosis.
Perhaps of importance to the biology of mechanisms involved in the regulation of plasma NOx levels is the detection of a minor residual additive genetic component to the phenotypic variance. This indicates the possible action of additional genes that directly influence quantitative variation in plasma levels of NOx. Such genes may be many with very small additive effects or this residual additive genetic component may subsume the effect of one or more loci with modest influence on plasma NOx levels. Increased family size, particularly by means of extending pedigrees, additional markers in the functional gene itself, and a better characterization of the interactions between this locus and relevant biological and environmental factors would improve our resolution of the contributions of the other quantitative trait loci in addition to that detected in this study.
Due to the radical nature of NO, which quickly oxidizes to NOx in vivo and in vitro, direct determination from plasma is difficult.16 17 18 19 20 However, studies have shown that measurement of NOx in blood collected after an overnight (12 to 14 hours) fast can reliably reflect basal (endogenous) NO production.20 23 24 Dietary nitrite/nitrate content could be a potential confounding factor for the plasma estimation, but we reduced the likelihood of the measurement's being affected significantly by the diet of previous days by collecting the blood after an overnight fast.20 24 While Jungersten et al25 suggested that oral intake of nitrate should be restricted for at least 48 hours, others indicated that dietary nitrates are eliminated from the blood by urinary excretion after approximately 12 to 16 hours.20 24 It should also be pointed out that a restricted diet may create an artificial condition and not reflect the normal physiological condition, which is more relevant to atherogenesis, a chronic process that may start from early life and last for decades. Although a low nitrite/nitrate diet was not employed for the present study, identification and modeling of dietary factors that actually influence variation in plasma NOx levels would only explain a greater proportion of the residual phenotypic variance and/or decrease the random environmental contribution to the variance in the phenotype. This would increase the relative signal of the quantitative trait locus effect detected in this family study and facilitate the detection and characterization of the genes that contribute to the residual additive genetic component of the variance in plasma NOx levels.
In conclusion, we report a major gene effect on plasma NOx levels, ie, NO production. The measured ecNOS4 polymorphism accounts for over 25% of the basal plasma NO production, indicating that the gene may contribute significantly to mechanisms mediating atherogenesis and other conditions.
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Selected Abbreviations and Acronyms |
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Appendix 1 |
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Statistical Analysis
Quantitative Genetic Analysis
According to classic quantitative genetic theory,26 the total phenotypic variance in a trait,
2P can be decomposed into the
variance due to the effects of genes,
2G, and the variance due to
the environmental effects, 2E.
These effects are additive, such that
 | (1) |
2G/2P.
For the initial detection of the effects of genes on phenotypic
variation in circulating NOx levels, we modeled the
NOx phenotype as
 | (2) |
 | (3) |
is the nxn matrix of kinship coefficients, 
is a
matrix in which the ijth element is the probability that the
ith and jth individuals share two genes IBD at
any given locus, and In is an identity matrix of order n.
The variance terms in equation 3
include the additive genetic variance,
the variance attributable to dominance effects, and the random
environmental variance. If multivariate normality is
assumed, the likelihood of the pedigree is easily calculated and
numerical optimization routines can be employed for the estimation of
parameter values.
On the basis of maximum-likelihood estimation theory, this approach
permits hypothesis testing as well as parameter estimation.
This is accomplished by comparing a general model, in which all
parameters are estimated, to one or more restricted models
in which the parameter estimate is constrained to zero or
some other value. Likelihood ratio test statistics are used to reject
less adequate models in comparisons between each restricted model and
the general model. The hypothesis to be tested is the null hypothesis
of no additive genetic variance for the NOx
phenotype (ie, h2=0). The likelihood ratio test
statistic. , is obtained as
 | (4) |
2 with a point mass at zero.27 In
all other comparisons, the likelihood ratio test statistic is
distributed asymptotically approximately as a 2
variate with degrees of freedom equal to the difference in the number
of parameters estimated in the two models.
We first conducted a univariate, quantitative genetic
analysis of circulating NOx levels in which
maximum-likelihood methods were used to simultaneously
estimate the phenotypic mean (µ), phenotypic standard deviation
(p), h2, and the effects of sex, age-by-sex,
and age2-by-sex. Using our modified version of
PAP,28 we maximized the likelihood of this model, in which
all the genetic variance was attributable to additive effects of genes,
on the data from 428 individuals from 108 pedigrees. We then maximized
the likelihood of an alternate, nested model in which h2
was constrained to equal zero. Comparison of the likelihoods of these
two models was made by means of a likelihood ratio test. Tests of the
significance of the sex and age terms in this model were conducted by
means of likelihood ratio tests as well.
Complex Segregation Analysis
To characterize further the genetic contribution to the
variance in plasma NOx levels we employed complex
segregation analysis29 to detect and measure the
contribution of a single locus (although not necessarily the only
locus) to plasma NOx using data from the families described
above. This approach entails the statistical comparison of the
likelihoods for alternate models (a nested subset of more restricted
nongenetic and genetic models, each representing different
transmission hypotheses for plasma concentrations of NOx)
with that of an unrestricted general model. The general transmission
model30 assumes a mixture of three normal phenotypic
distributions with a common SD and residual additive genetic
contribution to the phenotypic variance. The three phenotypic
distributions are referred to as "ousiotypes"31 and,
in the case of a segregating major locus, are interpreted to reflect
unobservable genotypes. Ousiotypes are a result of influence
from two discrete factors, A and/or a. In
ousiotype notation, upper-case letters indicate factors associated with
lower levels of the trait and lower-case letters indicate higher
levels. The expected relative frequencies of the three possible
ousiotypes (AA, Aa, and aa) are
assumed to conform to the classic Hardy-Weinberg proportions such that,
given a single parameter pA=p, the ousiotype
relative frequencies are predicted by
p2:2p(1-p):(1-p)2.
Under the mixed model for complex segregation analysis,
which includes a major factor and a residual additive genetic
component, the phenotype of the jth individual with
ousiotype i is
 | (5) |
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above). The genetic
component, 2g, of the conditional variance,
given immediately above, represents the residual additive
genetic variance. The unconditional variance of y has an additional
variance component attributable to the effect of the major factor and
is given by:
 | (6) |
AA,
Aa, and aa,). The parameters
estimated in the simplest of the general models for plasma levels of
NOx in this study were the frequency of the factor, or
allele in a genetic model, producing lower plasma NOx
levels; the mean of the three phenotypic distributions
(µAA, µAa, and µAA); an
additive genetic residual heritability (h2); a common
phenotypic SD (), assumed to be the same for each distribution;
three transmission probabilities (AA, Aa,
and aa,); plus the effects of sex and the linear and
quadratic age-by-sex terms (ßsex, ßagemales,
ßagefemales, ßage2 males, and
ßage2females).
We tested four classes of restricted models against the most general
model using the unified approach of Lalouel et al.30 The
simplest alternate model to be considered was the sporadic model, which
allows only random environmental effects and no genetic transmission
(h2=0). The second alternate model, the polygenic model,
assumes a single distribution to which the genetic contribution is
entirely due to the additive effects of genes. The third class of
alternate models, the Mendelian models, assumes the segregation of a
major locus effect and incorporates transmission probabilities fixed at
classical Mendelian expectations (ie, AA=1,
Aa=0.5, aa,=0). Additionally, Mendelian
mixture models allow for residual polygenic background. The fourth
model class tested, the environmental transmission model, assumes
random environmental effects for major factors with the transmission
probabilities constrained to equal pA. Environmental
mixture models permit the additional estimation of residual polygenic
inheritance.
Data on five additional variables (levels of apolipoprotein AI,
apolipoprotein B, creatinine, HDL cholesterol,
and total cholesterol), each of which were viewed as a
potential covariate in our analyses, were also available. We
used likelihood ratio tests to screen these five potential covariates
in the following manner. The likelihoods of two nested models, the
polygenic and codominant mixture models, that included a potential
covariate were compared by likelihood ratio test with those of the same
two nested models in which the regression parameter for
that covariate was fixed at 0. The tested covariate was retained for
estimation in the subsequent segregation analyses if the
likelihood ratio test statistic was significant at the 
=0.10 level.
It is analytically possible to simultaneously estimate the
effects of all five potential covariates in all the nested models.
However, because our statistical genetic approach uses only individuals
with complete data on all variables included in the maximized
models, and complete data on all five covariates were not available for
all individuals in our sample, we tested the significance of each
covariate singly in an attempt to retain the largest possible
proportion of the original 428 family members in the subsequent
segregation analysis.
Measured Genotype Approach
We used the "measured genotype"
approach27 32 to assess the effects of specific
alleles on variation in plasma NOx levels. In this
method, the mean effect of each allele is computed as the
difference in phenotype value between the population mean and
individuals carrying the allele. The contribution of the locus to
quantitative variation in the phenotype is the ratio of the
variance among the mean phenotype values for the separate
genotypes to the total phenotypic variance. When a
polymorphic marker identifies a functional mutation in a locus,
this ratio is a reasonable estimate of the contribution of that locus
to variation in the trait of interest. In the present case, where
the marker is located in intron 4 of the ecNOS4 gene, this contribution
generally will be less than the contribution of the associated
quantitative trait locus and reflective of gametic or linkage
disequilibrium with the functional locus.33
Using maximum-likelihood estimation techniques implemented in our
modified version of the computer program PAP,
v3.0,28 we conducted a measured genotype
analysis of plasma NOx levels in the members of
this sample who were genotyped at the ecNOS4 maker locus. The
parameters estimated in this analysis included the
frequency of the "low" plasma NOS allele (designated
pA), three genotypic means (µAA,
µAa, µaa), the residual phenotypic standard
deviation (), the residual additive genetic heritability
(h2), and the mean effects of sex (ßsex), age-by-sex
(ßagefemales; ßagemales), and
age2-by-sex (ßage2females;
ßage2 males). The hypothesis of no measured
gene effect was tested by means of a likelihood ratio test in which the
likelihood of a more general model in which three genotypic means were
estimated was compared with that of a restricted model in which the
genotypic means were constrained to be equal. The likelihood ratio test
statistic, , is usually obtained as two times the difference in the
ln likelihoods of the two models and is distributed approximately
asymptotically as a 2 variate with degrees of
freedom equal to the difference in the number of parameters
estimated in the two models.34 If a measured gene effect
is found to be significant, the total phenotypic covariance
matrix can be partitioned into its component covariance
matrices due to the measured gene, residual additive genes, and
residual environments35 to calculate the proportions of
the total covariance attributable to each.
Variance Component Linkage Analysis
To test for linkage between the ecNOS4 polymorphism and a
quantitative trait locus influencing variation in the plasma
NOx phenotype, we employed a general
variance-components linkage analysis developed by
Blangero36 and Blangero and Almasy.38 This
method is an extension of the strategy developed by Amos39
to estimate the genetic variance attributable to the region around a
specific marker locus. The approach entails specifying the expected
genetic covariances between arbitrary relatives as a function
of IBD relationships at a given marker locus that is assumed to be in
tight linkage with a quantitative trait locus. The variance component
method is more powerful than the widely used sibpair test of Haseman
and Elston40 because it can use information on all types
of relatives and provide an estimate of the relative variance of a
trait that is determined by an underlying major locus while allowing
for the simultaneous estimation of residual genetic
effects, covariate effects, and random environmental
effects.36
For the current application of the variance-component linkage method,
the sampling unit is the nuclear family, and the covariance
matrix for a given nuclear family is given by:
 | (7) |
is a matrix with elements (
mij)
that are estimates of the proportion of genes that individuals
i and j shared IBD at marker locus m that is
linked to a quantitative trait locus; 2m is
the additive genetic variance attributable to the marker locus; is
the nxn matrix kinship coefficients; 2g and
2e, respectively, are the proportions of the
residual phenotypic variance attributable to, respectively, additive
genetic and random environmental effects; and In is an
identity matrix of order n.
Variance components and covariate effects were estimated
simultaneously by maximum-likelihood techniques. A
likelihood function assuming a multivariate normal
density was numerically maximized to obtain parameter
estimates. In the most general model, the following
parameters were estimated: the phenotypic mean (µ); the
effects of (ie, regression coefficients for) sex (ßsex), age-by-sex
(ßagefemales; ßagemales), and
age2-by-sex (ßage2females;
ßage2 males); the phenotypic standard
deviation (); and the proportions of the residual phenotypic
variance attributable to the effects of residual additive genes
(h2), the marker locus that is linked to the quantitative
trait locus (h2m), and random environmental
effects (e2).
The hypothesis of no linkage is tested also by likelihood ratio tests that compare the likelihood of the general model in which h2m is estimated with that of an alternate, restricted model in which h2m was constrained to equal zero
Pedigree, phenotype, and genotype data for the statistical genetic analyses are managed and prepared for statistical genetic analysis using the computer program package PEDSYS.41 The variance-component linkage method itself is implemented by us using the computer program FISHER.42 This method requires an accurate estimate of the probability that alleles at the marker are IBD for pairs of relatives (in this analysis, nuclear family members). We employed the programs FSP and SIBPAL (SAGE 1994)43 to obtain maximum-likelihood estimates of the proportion of marker alleles at the ecNOS4 locus that are IBD for each relative pair.
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Acknowledgments |
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This work was supported by a grant from NH&MRC (940521) and an International Atherosclerosis Society Visiting Fellowship for X.L. Wang and D.E.L. Wilcken, and a grant from the US NIH (HL45522) for M.C. Mahaney, J. Blangero, and L. Almasy. We would like to thank Judy Lynch and the team members of the Heart Health Education Program for recruiting families in this study. Some of the results of the research reported in this manuscript were obtained using the program package SAGE, which is supported by a US Public Health Service Research source grant (1 P41 RR03655) from the Division of Research Resources.
Received December 13, 1996; accepted March 10, 1997.
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References |
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