721 Hutterites were genotyped for 65 biallelic polymorphisms in 36 genes using a linear array system developed by Roche Molecular Systems (Alameda, CA). These polymorphisms were selected as candidate markers for cardiovascular disease based on published reports. Allele frequencies are based on best-linear unbiased estimates that take into account the relatedness between individuals (McPeek et al. 2004). All polymorphisms are written with the higher frequency allele first.

Table 1. Allele Frequencies
 

Gene	Polymorphism	rs#	Location	Minor Allele Frequency	Standard Deviation
ACE	intron 16 ins/del	1799752	17q23	0.301	0.087
ADD1	gly460trp	4961	4p16.2	0.126	0.063
ADRB2	gly16arg	1042713	5q32	0.313	0.088
ADRB2	gln27glu	1042714	5q32	0.486	0.094
ADRB3	trp64arg	4994	8p11.22	0.027	0.031
AGT	thr235met	699	1q42.13	0.462	0.095
AGTR1	1166 A/C	5186	3q24	0.302	0.087
APOA4	thr347ser	675	11q23.3	0.351	0.091
APOA4	gln360his	5110	11q23.3	0	-
APOB	thr71ile	1367117	2p24	0.188	0.074
APOB	arg3500gln	5742904	2p24	0	-
APOC3	-641 C/A	2542052	11q23.3	0.485	0.095
APOC3	-482 C/T	2854117	11q23.3	0.279	0.085
APOC3	-455 T/C	2854116	11q23.3	0.483	0.095
APOC3	1100 C/T	4520	11q23.3	0.321	0.089
APOC3	3175 C/G	5128	11q23.3	0.139	0.066
APOC3	3206 T/G	4225	11q23.3	0.334	0.090
APOE	cys112arg	429358	19q13	0.139	0.066
APOE	arg158cys	7412	19q13	0.018	0.025
CBS	ile278thr,68-bp ins*	5742905	21q22.3	0.040	0.037
CETP	-631 C/A	1800776	16q21	0.006	0.015
CETP	-629 A/C	1800775	16q21	0.432	0.099
CETP	ile405val	5882	16q21	0.376	0.092
CETP	asp442gly	2303790	16q21	0	-
CETP	intron 14 G+1A	-	16q21	0	-
CETP	intron 14 +3 ins T	-	16q21	0	-
F2	20210 G/A	1799963	11p11.1	0	-
F5	arg506gln	6025	1q23	0.054	0.043
F7	arg353gln	6046	13q34	0.272	0.084
F7	-323 del/ins	5742910	13q34	0.274	0.085
FGB	-455 G/A	1800790	4q28	0.217	0.078
GNB3	825 C/T	5443	12p13	0.302	0.087
ICAM1	gly214arg	1799969	19p13	0.113	0.060
ITGA2	873 G/A	1062535	5q11.2	0.453	0.094
ITGB3	leu33pro	5918	17q21	0.174	0.072
LDLR	NcoI+/-	5742911	19p13.2	0.300	0.087
LIPC	-480 C/T	1800588	5q21	0.196	0.076
LPA	+93 C/T	1652503	6q27	0.123	0.063
LPA	+121 G/A	1800769	6q27	0.121	0.062
LPL	-93 T/G	1800590	8p22	0	-
LPL	asp9asn	1801177	8p22	0	-
LPL	asn291ser	268	8p22	0	-
LPL	ser447term	328	8p22	0.113	0.060
LTA	thr26asn	1041981	6p21.3	0.172	0.072
MMP3	-1171 A5/A6	3025058	11q22	0.329	0.090
MTHFR	677 C/T	1801133	1p36.3	0.390	0.093
NOS3	-922 A/G	1800779	7q36	0.469	0.094
NOS3	-690 C/T	3918226	7q36	0.076	0.051
NOS3	asp298glu	1799983	7q36	0.493	0.094
NPPA	664 G/A	5063	1p36.2	0	-
NPPA	2238 T/C	5065	1p36.2	0.065	0.047
PAI1	-675 G5/G4	1799768	7q22	0.368	0.092
PAI1	11053 T/G	7242	7q22	0.312	0.088
PON1	leu55met	3202100	7q21	0.252	0.083
PON1	gln192arg	662	7q21	0.318	0.088
PON2	ser311cys	7493	7q21	0.220	0.079
PPARG	pro12ala	1801282	3p25	0.139	0.066
SCNN1A	trp493arg	5742912	12p13	0.060	0.045
SCNN1A	ala663thr	2228576	12p13	0.327	0.089
SELE	leu554phe	5355	1q23	0	-
SELE	ser128arg	5361	1q23	0.054	0.043
TNF	-376 G/A	1800750	6p21.3	0.026	0.030
TNF	-308 G/A	1800629	6p21.3	0.025	0.029
TNF	-244 G/A	673	6p21.3	0	-
TNF	-238 G/A	361525	6p21.3	0.121	0.062

*Flanking sequence in this record is from cDNA. Linear array assay cannot distinguish between heterozygous and homozygous carriers of the insertion.
 
 

Tests of Association

To test for association with quantitative traits (triglycerides [TG], LDL, HDL, Lp(a), SBP, DBP), we used the general two-allele model (GTAM) test, which takes into account the relatedness between all individuals in our sample and assesses the fit of a quantitative trait to various genetic models (dominant, recessive, additive) (Abney et al. 2002). In these analyses, age and gender were included as covariates.

In addition, we tested these alleles for association with lipid levels and hypertension (HTN) using two case-control association tests (corrected chi-squared and quasi-likelihood) that take into account the relatedness between all the individuals in the sample (Bourgain et al. 2003). The cases were individuals in our sample in the highest quartile for LDL, Lp(a) and TG and the lowest quartile for HDL, while the controls were the individuals in the lowest quartile for LDL, Lp(a) and TG and the highest quartile for HDL, after adjusting for age and gender; the cases for HTN were taking antihypertensive medication, stage 1 or higher hypertension as defined by the World Health Organization-International Society of Hypertension (1999), or children with high blood pressure as defined by Williams et al. (2002) and the controls for HTN were age and gender matched normotensive individuals for all cases under age 40, plus all normotensive adults over age 40.

Lipid levels were determined by measurement of blood samples drawn after an overnight fast. All associations with p <0.05 for at least one test of association are included in tables 2-6. For the GTAM results, direction indicates whether increasing (+) or decreasing (-) phenotypic values were associated with an increasing number of copies of the indicated allele. For the case-control tests, the allele that has higher frequency in the cases is listed. Abbreviations: GTAM = general two-allele model, na = not applicable, HT = heterozygote, Chi² = corrected chi-squared method, QL = quasi-likelihood method, SBP = systolic blood pressure, DBP = diastolic blood pressure, HTN = hypertension

Table 2. Serum Triglycerides (TG) Levels. TG was measured on 485 Hutterites age 14 and older.
 

		GTAM Results				Case:Control Results
Gene	Polymorphism	Allele	Direction	Model	p-value	Allele	Chi2 p-value	QL p-value
APOC3	-641 A/C	A	+	dominant	0.010	A	0.078	0.088
APOC3	-482 C/T	C	-	additive/recessive	0.016	C	0.040	>0.10
APOC3	-455 T/C	T	-	additive/recessive	0.013	C	0.063	>0.10
APOC3	1100 C/T	C	-	additive	>0.10	T	>0.10	0.036
APOC3	3175 C/G	G	+	dominant	0.000093	G	0.0063	0.000016
APOC3	3206 T/G	G	+	dominant	0.047	G	>0.10	0.0065
CETP	-631/-629 haplotype	CA	-	recessive	0.045	CC	>0.10	>0.10
ICAM1	gly214arg	arg	+	additive	0.032	arg	0.0054	0.096
PON2	ser311cys	na	na	HT lowest	>0.10	ser	0.042	>0.10
SCNN1A	ala663thr	ala	+	dominant	0.048	ala	>0.10	>0.10
SELE	ser128arg	arg	+	additive	>0.10	arg	0.054	>0.10

 

Table 3. Serum LDL Levels. LDL-cholesterol levels were determined for 452 Hutterites age 14 and older.
 

		GTAM Results				Case:Control Results
Gene	Polymorphism	Allele	Direction	Model	p-value	Allele	Chi2 p-value	QL p-value
AGTR1	1166 A/C	C	-	recessive	0.0064	A	0.0025	0.013
APOC3	1100 C/T	T	-	additive	>0.10	C	0.042	>0.10
APOC3	3206 T/G	G	-	additive	>0.10	T	0.026	>0.10
APOE	haplotype	E2	-	additive	0.0010	E4	0.057	0.000060
FGB	-455 G/A	Na	na	HT highest	>0.10	A	>0.10	0.025
LDLR	NcoI+/- (A/G)	-	-	additive	0.063	+	0.0045	0.00022
LTA	thr26asn	na	na	HT lowest	0.0081	thr	0.012	0.0070
NPPA	2238 T/C	Na	na	HT lowest	>0.10	T	0.039	>0.10

 

Table 4. Serum HDL Levels. HDL-cholesterol levels were measured on 485 Hutterites age 14 and older.
 

		GTAM Results				Case:Control Results
Gene	Polymorphism	Allele	Direction	Model	p-value	Allele	Chi2 p-value	QL p-value
AGTR1	1166 A/C	C	+	additive	>0.10	A	0.032	0.096
APOC3	-641 A/C	na	na	HT lowest	0.023	A	>0.10	>0.10
APOC3	-455 T/C	na	na	HT lowest	0.027	C	>0.10	>0.10
APOC3	3175 C/G	na	na	HT lowest	>0.10	G	0.078	0.042
CETP	-631/-629 haplotype	CA	+	additive	0.000013	CC	0.00015	0.000039
ICAM1	gly214arg	gly	+	additive/recessive	>0.10	arg	0.0056	>0.10
PON2	ser311cys	na	na	HT highest	0.021	ser	0.0091	0.020
SCNN1A	trp493arg	arg	-	additive	0.058	arg	0.021	0.070
SELE	ser128arg	arg	-	additive	0.041	arg	0.037	0.019

 

Table 5. Serum Lipoprotein(a) Levels. Lp(a) levels were determined for 374 Hutterites age 14 and older.
 

		GTAM Results				Case:Control Results
Gene	Polymorphism	Allele	Direction	Model	p-value	Allele	Chi2 p-value	QL p-value
APOB	thr71ile	thr	-	additive/recessive	0.019	ile	>0.10	0.026
CETP	ile405val	val	+	additive/recessive	0.048	val	>0.10	>0.10
F7	arg353gln	arg	-	additive	0.079	gln	0.012	0.086
F7	-323 del/ins	ins	+	additive	0.067	ins	0.0037	0.027
FGB	-455 G/A	A	-	additive	0.043	G	0.013	0.039
ITGB3	leu33pro	pro	+	dominant	>0.10	pro	0.011	>0.10
LPA	93 C/T	T	+	additive	0.000000029	T	0.074	0.00029
NOS3	-690 C/T	T	-	additive	>0.10	A	0.038	0.070
SELE	ser128arg	ser	+	dominant	0.038	arg	0.027	>0.10

 

Table 6. Blood Pressure. Blood pressure was measured on 623 Hutterites age 5 and older.
 

GTAM Results (SBP)

GTAM Results (DBP)

Case:Control Results (HTN)

Gene

Polymorphism

Allele

Direction

Model

p-value

Allele

Direction

Model

p-value

Allele

Chi2 p-value

QL p-value

ACE

intron 16 ins/del

ins

+

additive

0.0026

ins

+

additive

>0.10

del

>0.10

>0.10

AGT

thr235met

met

-

additive

0.027

met

-

additive

0.037

thr

0.050

>0.10

FGB

-455 G/A

G

+

additive

0.015

A

-

additive

0.058

G

>0.10

>0.10

LIPC

-480 C/T

na

na

HT highest

>0.10

T

+

additive

0.0022

T

0.0048

0.0090

LTA

thr26asn

asn

+

additive/recessive

>0.10

asn

+

recessive

0.039

asn

0.011

0.033

PON2

ser311cys

na

na

HT lowest

0.042

na

na

HT lowest

>0.10

ser

>0.10

>0.10

TNF

-308 G/A

G

-

additive

>0.10

G

-

additive

0.074

A

0.030

0.013

 

References