Patent Application
20080015141
A correlation study, in which the genotype of the hsgk1 gene in different patients (twins) was compared with the systolic and diastolic blood pressure values which were measured in these patients, and then statically evaluated, was carried out within the context of the present invention.
75 dizygotic pairs of twins were used for the correlation analysis (Busjahn et al., J. Hypertens 1996, 14: 1195-1199; Busjahn et al., Hypertension 1997, 29: 165-170). The experimental subjects all belonged to the German-Caucasian race and originated from different parts of Germany. Blood was taken from the pairs of twins, and from their parents, for the purpose of verifying the dizygotism and for further molecular genetic analyses. Each of the experimental subjects underwent a prior medical examination. None of the experimental subjects was known to be suffering from any chronic medically recognized disease. After 5 min, the blood pressure of the test subject, whose was in the sitting position, was measured by a trained physician using a standardized mercury sphygmomanometer (2 measurements at a time interval of 1 min). The mean of the two measurements was used as the blood pressure value.
The advantage of using dizygotic twins for correlation studies is that they are of the same age and that the external influences on their phenotypes are to be judged as being minimal (Martin et al., Nat Genet 1997, 17: 387-392).
The importance of studies on twins for the elucidation of complex genetic diseases was recently described by Martin et al., 1997.
The dizygotism of the pairs of twins was confirmed by using the polymerase chain reaction (PCR) to amplify five microsatellite markers. In this analysis of microsatellite markers, deoxyribonucleic acid (DNA) fragments are amplified by PCR using specific oligonucleotides which contain regions which are highly variable in different human individuals. The high degree of variability in these regions of the genome can be detected by means of slight differences in sizes of the amplified fragments, resulting, when there is diversity at the corresponding gene locus, in double bands, i.e. what are termed microsatellite bands, being formed after the PCR products have been subjected to gel-electrophoretic fractionation (Becket et al., J. Reproductive Med 1997, 42: 260-266).
For the purpose of carrying out a molecular genetic analysis of the target gene, in the present case the hsgk1 gene, three further microsatellite marker regions (d6s472, d6s1038, d6s270) in the immediate vicinity of the hsgk1 locus were amplified by PCR and then compared with the corresponding samples from the other twin and the parents. In this way, it was possible to decide whether the twins had inherited alleles, from their parents, which were identical or different with regard to the allele under investigation. The correlation analysis was carried out using the structural equation modelling (SEM) model (Eaves et al., Behav Genet 1996, 26: 519-525; Neale, 1997: Mx: Statistical modeling, Box 126 MCV, Richmond, Va. 23298; Department of Psychiatry. 4th edition). This model is based on variance-covariances matrices of the test pairs which are characterized by the probability that they possess either no, one or two identical alleles. The variance with regard to the phenotype was divided into a variance which is based on the genetic background of all the genes (A), a variance which is based on the genetic background of the target gene (Q), in this case the hsgk1 gene, and the variance due to external influences (E).
VAR=A
2
+Q
2
+E
2
The covariance of a test pair was defined for the three possible allele combinations IBD0, IBD1 and IBD2 (IBD=identical by descent; 0, 1 or 2 identical alleles) as follows:
COV(IBD0)=0.5A2
COV(IBD1)=0.5A2+0.5Q2
COV(IBD2)=0.5A2+Q2
In order to evaluate the correlation between the genetic makeup of the hsgk1 locus and the blood pressure of the test subject, the differences between models which do and, respectively, do not take into account the genetic variance with regard to the target gene hsgk1 were calculated as χ2 statistic. For each pair and each gene locus, the allele ratios were calculated by means of the so-called multipoint model (MAPMAKER/SIBS; Kruglyak et al., Am J Hum Genet 1995, 57: 439-454) based on the parental genotypes.
The greater informative value of the analytical method which is based on a variance-covariance evaluation, as compared with the above-described χ2 statistic (S.A.G.E. Statistical Analysis for Genetic Epidemiology, Release 2.2. Computer program package, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, Ohio, USA, 1996) was recently confirmed in a simulation study (Fulker et al., Behav Gen 1996, 26: 527-532). An error probability of p<0.01 was accepted in order to ensure a significant correlation with regard to the criteria of Lander and Kruglyak (Lander et al., Nat Genet 1995, 11: 241-246).
Table 1 shows the results of this correlation study,
As can be seen from Table 1, the low values for the ascertained error probabilities p, which do not exceed, or only slightly exceed, the accepted error probability of p<0.01, prove that there is a direct correlation between the genetic variance with regard to the hsgk1 gene locus and the phenotypically ascertained variance of the measured blood pressure.
The genomic organization of the hsgk1 gene has already been described (Waldegger et al., Genomics, 51, 299 [1998]), http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000118515).
In order to identify SNPs whose occurrences are relevant for a predisposition for developing hypertension, the SNPs in the hsgk1 gene which were published in databases were first of all investigated in order to determine whether they are genuine SNPs, and not simple sequencing errors, and whether the SNPs are sufficiently polymorphic in order to form the basis for a diagnostic detection of a predisposition for hypertension. The SNP rs 1057293 in exon 8, which concerns the replacement of a C with a T (http://www.ensembl.org/Homo_sapiens/snpview?snp=1057293; http://www.ncbi.nln.nih.gov/SNP/snp_ref.cgi?type=rs&rs=1057293) and a second SNP, which is located in the hsgk1 gene, at a distance of precisely 551 bp from the first SNP, in the donor splice site of intron 6 to exon 7 and concerns the replacement of a T with a C, had already been located in this way.
Blood samples were taken from a random sample of the 75 pairs of twins. After the genomic DNA of the hsgk1 gene had been amplified from the blood samples by means of PCR, the exons and introns (but not the promoter region) of the hsgk1 gene were sequenced directly and completely using suitable sequencing priers. When the sequences of the hsgk1 genes which originated from different test subjects were compared, a further polymorphism in intron 2, consisting of the insertion of an additional nucleotide G in position 732/733, was noted. Furthermore, the presence or absence of this G insertion at position 732/733 in the hsgk1 genes of the individual test subjects exhibited a significant correlation with the blood pressure which was measured in the individual test subjects: on average, InsG/InsG genotypes exhibited significantly lower systolic and diastolic blood pressure values that did the less frequent WT/WT genotypes as well as the heterozygous WT/InsG genotypes (see Table 3). By contrast, other polymorphisms in the hsgk1 gene exhibited a correlation with the measured blood pressure which was less significant (e.g. intron 6 (C2071T) and exon 8 (T2617C, D240D)) or else no correlation with the measured blood pressure (e.g. intron 3 position Ins 13+xT, T1300-1312 and intron 4 (C1451T) and intron 7 position 2544delA), as Table 2 shows.
The ECG values, which were likewise measured on the test subjects, also showed that there was a marked correlation of the Q/T intervals, which were determined for the individual test subjects, with the genotype of the test subjects with regard to the polymorphism in intron 2 at position 732/733 of the hsgk1 gene: in this connection, test subjects possessing the less frequent WT/WT genotype exhibited markedly shorter Q/T intervals than heterozygous WT/InsG test subjects, while these latter in turn exhibited significantly shorter Q/T intervals than did test subjects possessing the more frequent InsG/InsG genotype (see Table 3). Longer Q/T intervals increase the danger of contracting cardiac rhythm disturbances, such as, in particular, the long Q/T syndrome. Consequently, inverse correlations are found between the genotype of the polymorphism in intron 2 at position 732/733 of the hsgk1 gene and a predisposition for the long Q/T syndrome, on the one hand, and a predisposition for hypertension, on the other hand. These correlations can in each case be used for the diagnosis, therapy and prophylaxes of hypertension and the long Q/T syndrome.
| Number | Date | Country | Kind |
|---|---|---|---|
| 103 05 213.5 | Feb 2003 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/01051 | 2/5/2004 | WO | 00 | 3/8/2006 |
