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- TDT, transmission disequilibrium test
- S-TDT, sibship transmission disequilibrium test
- RAS, renin angiotensin system
Hypertension is one of the most important risk factors for cardiovascular diseases. Despite extensive research examining the causes of blood pressure variation, a significant proportion of blood pressure variation is yet to be explained. Studies of families and twins suggest that 20-40% of blood pressure variation can be attributed to genetic factors.1 Evidence shows that the genetic contribution is even greater for young onset hypertension.2 We feel that genetic approaches focusing on young onset hypertension will provide new insight into the pathogenesis of hypertension.
In our previous report, the affected sib pairs (25 independent, affected sib pairs) method showed positive signs of linkage for markers of the atrial natriuretic peptide gene (NPPA) (D1S1612, p=0.0162), angiotensinogen gene (AGT) (D1S547, p=0.0263), lipoprotein lipase gene (LPL) (D8S1145, p=0.0284), and angiotensin converting enzyme gene (DCP1) (D17S2193, p=0.0256),3 indicating that multiple pathogenic pathways may be involved in the aetiology of young onset hypertension. Owing to this aetiological complexity, in the current study we focus on high resolution mapping of AGT (located on 1q42-43) and DCP1 (located on 17q23), genes of the renin angiotensin system (RAS). Renin catalyses the first step of the activation pathway of angiotensinogen to angiotensin I, which is then cleaved to angiotensin II by angiotensin I converting enzyme. This cascade can lead to aldosterone release, vasoconstriction, and increased blood pressure. Although the RAS has been extensively studied, it remains unclear how and to what extent RAS gene variants contribute to the blood pressure variations in various human populations.
MATERIALS AND METHODS
We have recruited 59 nuclear families (a total of 214 subjects) from a hypertension clinic at Taipei Veterans General Hospital, Taiwan. Our study group included 81 young onset hypertensive patients (59 probands and 22 affected sibs, mean age 30.4 (SD 0.95)), 39 normotensive sibs (mean age 32.2 (SD 1.6)), and 94 parents. Our previous study included 25 affected sib pairs from 18 families for affected sib pair analysis. This transmission disequilibrium test (TDT) study used information from all 59 families with probands. Therefore, the former is a subset of the latter. The protocol of this study was approved by the Human Investigation Committee of the Institute of Biomedical Sciences, Academia Sinica.
Polymorphic microsatellite markers located on 1q42-43 and 17q23 were selected based on GeneMap'99 and comprehensive human genetic maps from the Marshfield Medical Research Foundation, and obtained from Multi-Colored Fluorescent Human MapPairs Markers of Research Genetics (Huntsville, AL). Nine markers on 1q42-43 were selected: D1S2805 (245.05 cM), D1S3462 (247.23 cM), D1S459 (247.23 cM), D1S1540 (252.12 cM), D1S235 (254.64 cM), D1S517 (262.96 cM), D1S1149 (262.96 cM), D1S1594 (265.49 cM), and D1S547 (267.51 cM). The six markers on 17q23 were D17S1297 (83.40 cM), D17S1295 (83.40 cM), D17S942 (85.94 cM), ATA108a05 (88.76 cM), D17S789 (89.32 cM), and D17S2193 (89.32 cM). The polymerase chain reaction protocol for microsatellite markers was performed as previously reported.3 Fragment analysis was performed using an ABI 377 DNA sequencer and analysed by GeneScan version 3.0 and GenotypeR version 3.0. The allele calling was conducted independently by two readers and cross checked.
In addition to conventional TDT that only used data from heterozygous parents, an extended TDT (S-TDT and combined Z score) developed by Spielman et al4 was also carried out. Information from 42 young hypertensive patients and both of their parents (representing 35 families) were used for conventional TDT. In the sibship transmission disequilibrium test (S-TDT), information from 25 hypertensive patients and 21 normotensive sibs from 17 families were used. Then a Z score was obtained by combining information from the conventional TDT and the S-TDT. For comparisons, we also carried out Horvath's SDT,5 which uses an exact p value to test the difference between affected and unaffected sibs. In this test, only information from 44 young hypertensive patients and their normotensive sibs (39) were used. Since these markers were close to each other, we performed haplotype TDT using the “TRANSMIT” program.6 In this analysis, data from all 59 families were used. Information from the 24 single parent families was incorporated into the analysis by use of expectation maximisation algorithms. Because of our limited sample size, only haplotypes created by two markers were included.
Because we tested 15 markers with multiple alleles, a Bonferroni procedure was carried out to adjust for multiple comparisons, as suggested by Spielman et al.4
RESULTS AND DISCUSSION
No association with any marker in the region of 1q42-43 was found (data not shown). The AGT was the first gene linked to hypertension,7 but results showing linkage of AGT and hypertension has not been consistent across various populations. A meta-analysis concluded that AGT contributes significantly but moderately to human blood pressure variance.8 Our result is consistent with the negative findings of a study carried out in central China.9 However, a case-control study carried out by Chiang et al10 in Taiwan showed a positive association between the M235T polymorphism and hypertension in adults aged 60 and above.10 These divergent results may be the result of a different definition of hypertension and different research designs. Further studies are required to examine the role of the AGT gene in the pathogenesis of hypertension in Chinese as well as in other ethnic groups.
Association between allele 3 of ATA108a05 and young onset hypertension was shown by Spielman's (p<10-5) and Horvath's (p=0.027) TDTs (table 1). A weaker but similar association was observed between allele 4 of D17S789 and young onset hypertension using Spielman's (p=0.015) but not Horvath's method (p=0.197) (table 1). Because allele 3 of ATA108a05 shows strong association by both the Spielman and Horvath tests, our haplotype analysis was carried out focusing on this particular allele. In the two marker haplotype TDT analysis, four out of six estimated haplotypes that contain allele 3 of ATA108a05 and markers proximal or distal to it showed significant association after Bonferroni's correction (table 2). The insignificant associations were with the lower frequency alleles: allele 3 (4.4%) of D17S942 and allele 6 (1.6%) of D17S789. The results in tables 1 and 2 suggest that allele 3 of ATA108a05 warrants further investigation.
The insertion or deletion of a 287 bp Alu repeat element in intron 16 of the DCP1 gene has also been associated with coronary artery disease,11 but direct linkage between hypertension and DCP1 is preliminary.12 Many studies have shown that insertion/deletion (I/D) polymorphism of DCP1 is associated with hypertension,13,14 but none has shown linkage between hypertension and I/D polymorphism and other markers of DCP1. Two studies, using the quantitative trait locus approach, showed linkage between I/D polymorphism and blood pressure in a sex and age specific manner.14,15 A recent genome scan quantitative locus approach study by Levy et al16 found a linkage between systolic blood pressure and two peaks located on chromosome 17 at 67 cM and at 94 cM, and a linkage between diastolic blood pressure and a peak located on chromosome 17 at 74 cM. The DCP1 gene is located in the region from 85 cM to 90 cM. In our study, the region from 85.9 cM to 89.32 cM was strongly associated with hypertension. Our findings suggest that the marker (as close as 3 cM distal to DCP1) may be linked to young onset hypertension in Han Chinese.
Further studies are required to examine whether variation in DCP1 may contribute to young onset hypertension, considering that there are many functional genes and expressed sequence tags (ESTs) in the region near the DCP1 locus. Examples include growth hormone, voltage gated sodium channel type IV (SCN4A), and regulator of G protein signalling 9 (RGS9). Ascertainment of more probands with young onset hypertension in many clinics is in progress to replicate our finding.
This project was supported by the Frontier Program on Medical Gene Research, which was funded by the Department of Health (Grant DOH89-TD-1129) and by the National Science Council (NSC88-2318-B-001-010-M51) in Taiwan. We thank Jim Chen for his technical support in computer programming.
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