Novel polymorphic AluYb8 insertion in the WNK1 gene is associated with blood pressure variation in Europeans

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. Hum Mutat 32:1–9, 2011. © 2011 Wiley-Liss, Inc.

DHPLC primers were designed according to manufacturer recommendations (DHPLC; Wave Technologies Inc. USA) using web-based program Primer3 (3). The calculations of the appropriate melting conditions for each of the selected DHPLC PCR fragments we used web based DHPLC Melt Program (http://insertion.stanford.edu/melt.html). The uniqueness of the primer sequence in the human genome were tested and verifying using GenomeTester1.3 (http://bioinfo.ut.ee/genometester/). The DGGE and DHPLC primers are given in Supp. Table   S1.
Regions targeted for polymorphism screening were amplified by touchdown method using 100 ng genomic DNA, Smart-Taq Hot DNA polymerase (Naxo OÜ, Tartu, Estonia), GeneAmp PCR System 2700 thermal cycler (Applied Biosystems Inc., USA) and amplification conditions described elsewhere (1). DGGE electrophoresis by INGENY PhorU2 system was performed according to the manufacturer's instructions (Ingeny, Goes, Netherlands). Electrophoresis (16)(17) hours) was carried out at 58ºC, amperage of 40 mA and voltage of 100 V. The DHPLC analysis was performed according to the manufacturer's recommendations (Wave Technologies Inc. USA) using pools consisting of the genomic DNA from three individuals. Individual DNA probes exhibiting unusual pattern in the DGGE analysis or forming a DNA-pool indicative to the presence of a polymorphism in the DHPLC analysis were resequenced to determine the exact changes in the genomic sequence.

Recruitment of individuals to the HYPEST study
The HYPEST (n=1823) study has been approved by the Ethics Committee on Human Research of University of Tartu, Estonia. All of the study participants are of Eastern European ancestry, have filled a self-administrated epidemiological questionnaire recording to their past and present health and life-style and have given their written informed consent.
The HYPEST sample collection represents a case-cohort study. Essential hypertension patients were recruited by blood pressure specialists during the patients' ambulatory visits or hospitalization at the North Estonia Medical Center, Centre of Cardiology, or at the Cardiology Clinic, Tartu University Hospital, Estonia. The HYPEST healthy control cohort was recruited from among the long-term blood donors across Estonia. All the included donors had no personal history of cardiovascular diseases and had also never been prescribed any relevant medications.

Details of the cDNA synthesis protocol
Total RNA from leucocytes was extracted using LeukoLOCK™ Total RNA Isolation System (Ambion Inc, Austin, Texas, USA) including an optional TURBO™ DNase treatment to degrade genomic DNA. Concentration of extracted RNA was measured with NanoDrop® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, LLC, Wilmington, Delaware, USA).
RNA was reverse transcribed using SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Life Technologies Corporation, Carlsbad, California, USA) in the final volume of 20 µl containing 0,4 µg of RNA, 10 µl of 2X RT Reaction Mix [oligo(dT) 20 (2.5 μM), random hexamers (2.5 ng/μl), 10 mM MgCl2, and dNTPs], 2µl of RT Enzyme Mix (includes SuperScript™ III RT and RNaseOUT™) and DEPC-treated water. The reaction mixture was incubated at 25ºC for 10 minutes and at 50ºC for 30 minutes. Reaction was terminated at 85ºC at 5 minutes and chilled on ice. cDNA was treated with 2 U of E.coli Rnase H at 37ºC for 20 minutes to remove the RNA template from the cDNA:RNA hybrid molecule.
Supp. Figure S1. PCR amplification of the locus for WNK1 intron 10 AluYb8 insertion in eleven chimpanzees as resolved by agarose gel (3%) electrophoresis. In all analyzed individuals (numbered 1-11) the resulting PCR product corresponded to the wild-type genotype lacking the WNK1 intron 10 AluYb8 insertion. Human genomic DNAs representing alternative genotypes were used as positive controls: wild-type genotype without AluYb8 insertion (-/-, PCR product 353 bp); heterozygous (A/-) and homozygous (A/A, PCR product 660 bp) carriers of the insertion. N, negative control; M, marker (Fermentas MassRuler TM DNA Ladder, Low Range) Supp. Figure S2. Sequence alignment of the WNK1 genomic fragment spanning from exon 10 to exon 11. Human chromosomes carrying (Alu +) and not carrying (Alu -) the AluYb8 insertion are shown in comparison with the respective genomic region in common chimpanzee (Pan troglodytes). The sequences of the WNK1 exons 10 and 11 are shown in blue and the AluYb8 insertion to human WNK1 intron 10 in red font. The substitution-based divergence between human wild-type (Alu -) and chimpanzee sequences (excluding species-specific insertion/deletions) is 0%, 0.2% and 1.1% for exon 10 (150 bp), exon 11 (459 bp) and intron 10 (1211 bp), respectively. Sequence alignment was performed using the web-based global alignment program ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/).   The association testing was performed using the BRIGHT normotensive individuals (n=1421) genotyped in the current study for the AluYb8 insertion, and in the previous study for rs11064527, rs12816718, rs956868 (Newhouse et al., 2009). Statistical analysis was identical to the conditions described in Materials and Methods for the association testing with quantitative variables, SBP and DBP (linear regression, additive model, age and gender as covariates). SBP, DBP, systolic and diastolic blood pressure; SE, standard error