Katrin Kepp

The outcome of Genome-Wide Association Studies (GWAS) has challenged the field of blood pressure (BP) genetics as previous candidate genes have not been among the top loci in these scans. We used Affymetrix 500K genotyping data of KORA S3 cohort (n = 1,644; Southern-Germany) to address (i) SNP coverage in 160 BP candidate genes; (ii) the evidence for associations with BP traits in genome-wide and replication data, and haplotype analysis. In total, 160 gene regions (genic region+/-10 kb) covered 2,411 SNPs across 11.4 Mb. Marker densities in genes varied from 0 (n = 11) to 0.6 SNPs/kb. On average 52.5% of the HAPMAP SNPs per gene were captured. No evidence for association with BP was obtained for 1,449 tested SNPs. Considerable associations (P<10(-3)) were detected for the genes, where >50% of HAPMAP SNPs were tagged. In general, genes with higher marker density (>0.2 SNPs/kb) revealed a better chance to reach close to significance associations. Although, none of the detected P-values remained significant after Bonferroni correction (P<0.05/2319, P<2.15 x 10(-5)), the strength of some detected associations was close to this level: rs10889553 (LEPR) and systolic BP (SBP) (P = 4.5 x 10(-5)) as well as rs10954174 (LEP) and diastolic BP (DBP) (P = 5.20 x 10(-5)). In total, 12 markers in 7 genes (ADRA2A, LEP, LEPR, PTGER3, SLC2A1, SLC4A2, SLC8A1) revealed considerable association (P<10(-3)) either with SBP, DBP, and/or hypertension (HYP). None of these were confirmed in replication samples (KORA S4, HYPEST, BRIGHT). However, supportive evidence for the association of rs10889553 (LEPR) and rs11195419 (ADRA2A) with BP was obtained in meta-analysis across samples stratified either by body mass index, smoking or alcohol consumption. Haplotype analysis highlighted LEPR and PTGER3. In conclusion, the lack of associations in BP candidate genes may be attributed to inadequate marker coverage on the genome-wide arrays, small phenotypic effects of the loci and/or complex interaction with life-style and metabolic parameters.

BACKGROUND: Mammalian transcriptome contains a large proportion of diverse and structurally complex noncoding RNAs. One class of such RNAs, natural antisense transcripts (NATs), are derived from the opposite strand of many protein-coding genes. Although the exact structure and functional relevance of most of the NATs is unknown, their emerging role as gene expression regulators raises the hypothesis that NATs might contribute to development of complex human disorders. The goal of our study was to investigate the involvement of NATs in regulation of candidate genes for blood pressure. RESULTS: First we analysed blood pressure candidate genes for the presence of natural antisense transcripts. In silico analysis revealed that seven genes (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2) have an antisense partner transcribed from the opposite strand. We characterized NPPA and its antisense transcript (NPPA-AS) in more detail. We found that NPPA-AS is expressed in a number of human tissues as a collection of alternatively spliced isoforms and that NPPA-AS and NPPA can form RNA duplexes in vivo. We also demonstrated that a specific NPPA-AS isoform is capable of down-regulating the intron-retained NPPA mRNA variant. We studied the evolutionary conservation of NPPA-AS and were able to detect the presence of Nppa-as transcript in mouse. CONCLUSION: Our results demonstrate functional interaction of NPPA-AS with NPPA at the RNA level and suggest that antisense transcription might be an important post-transcriptional mechanism modulating NPPA expression.

BACKGROUND: Kidneys have an important function in blood pressure (BP) regulation and elevated BP may lead to kidney failure. Chr2p12-p13 region linked to BP traits in multiple studies harbours a potential candidate for BP and renal function, N-acetyltransferase 8 (NAT8) expressed in embryonic and adult kidney and associated with nephrotoxicity response. METHODS/RESULTS: We report the first study exploring NAT8 as a potential candidate gene for blood pressure and kidney function. The resequencing (n = 42, random Estonian samples) identified 15 NAT8 polymorphisms, including 6 novel variants. The diversity of NAT8 5' upstream region (pi/bp = 0.00320) exceeded up to 10 times the variation in the NAT8 genic region (pi/bp = 0.00037) as well as the average variation (pi/bp = 0.00040) for the promoters of 29 reference genes associated with hypertension. We suggest that a potential source for such high variation could be an active gene conversion process from NAT8B duplicate gene to NAT8. Similarly to NAT8, several reference genes with the most variable upstream regions have also duplicate copies. The NAT8 promoter SNPs were targeted with pilot quantitative association studies for blood pressure (n = 137, healthy unrelated individuals) and for the index of kidney function - estimated glomerular filtration rate (eGFR; n = 157 hypertensives with and without nephropathy). Minor alleles of these polymorphisms revealed a significant protective effect against elevated systolic BP as well as kidney failure in hypertension patients (p < 0.05; linear regression model, addictive effect). CONCLUSION: The full resequencing and pilot association study of a novel positional candidate gene for blood pressure and renal function, human N-acetyltransferase 8, suggested a contribution of highly variable NAT8 promoter polymorphisms in determination of systolic blood pressure and eGFR. Based on in silico analysis, we raise the hypothesis that the alternative SNP alleles of the NAT8 upstream region may have differential effect on gene expression.

Mutations in WNK1 and WNK4 cause familial hypertension, the Gordon syndrome. WNK1 and WNK4 conserved noncoding regions were targeted to polymorphism screening using DHPLC and DGGE. The scan identified an undescribed polymorphic AluYb8 insertion in WNK1 intron 10. Screening in primates revealed that this Alu-insertion has probably occurred in human lineage. Genotyping in 18 populations from Europe, Asia, and Africa (n = 854) indicated an expansion of the WNK1 AluYb8 bearing chromosomes out of Africa. The allele frequency in Sub-Saharan Africa was ~3.3 times lower than in other populations (4.8 vs. 15.8%; P = 9.7 × 10(-9) ). Meta-analysis across three European sample sets (n = 3,494; HYPEST, Estonians; BRIGHT, the British; CADCZ, Czech) detected significant association of the WNK1 AluYb8 insertion with blood pressure (BP; systolic BP, P = 4.03 × 10(-3) , effect 1.12; diastolic BP, P = 1.21 × 10(-2) , effect 0.67). Gender-stratified analysis revealed that this effect might be female-specific (n = 2,088; SBP, P = 1.99 × 10(-3) , effect 1.59; DBP P = 3.64 × 10(-4) , effect 1.23; resistant to Bonferroni correction), whereas no statistical support was identified for the association with male BP (n = 1,406). In leucocytes, the expressional proportions of the full-length WNK1 transcript and the splice-form skipping exon 11 were significantly shifted in AluYb8 carriers compared to noncarriers. The WNK1 AluYb8 insertion might affect human BP via altering the profile of alternatively spliced transcripts.

To survey the quality of SNP genotyping, a joint Nordic quality assessment (QA) round was organized between 11 laboratories in the Nordic and Baltic countries. The QA round involved blinded genotyping of 47 DNA samples for 18 or six randomly selected SNPs. The methods used by the participating laboratories included all major platforms for small- to medium-size SNP genotyping. The laboratories used their standard procedures for SNP assay design, genotyping, and quality control. Based on the joint results from all laboratories, a consensus genotype for each DNA sample and SNP was determined by the coordinator of the survey, and the results from each laboratory were compared to this genotype. The overall genotyping accuracy achieved in the survey was excellent. Six laboratories delivered genotype data that were in full agreement with the consensus genotype. The average accuracy per SNP varied from 99.1 to 100% between the laboratories, and it was frequently 100% for the majority of the assays for which SNP genotypes were reported. Lessons from the survey are that special attention should be given to the quality of the DNA samples prior to genotyping, and that a conservative approach for calling the genotypes should be used to achieve a high accuracy.