Siim Sõber

Genome-wide scan identifies CDH13 as a novel susceptibility locus contributing to blood pressure determination in two European populations

Hypertension is a complex disease that affects a large proportion of adult population. Although approximately half of the inter-individual variance in blood pressure (BP) level is heritable, identification of genes responsible for its regulation has remained challenging. Genome-wide association study (GWAS) is a novel approach to search for genetic variants contributing to complex diseases. We conducted GWAS for three BP traits [systolic and diastolic blood pressure (SBP and DBP); hypertension (HYP)] in the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) S3 cohort (n = 1644) recruited from general population in Southern Germany. GWAS with 395 912 single nucleotide polymorphisms (SNPs) identified an association between BP traits and a common variant rs11646213 (T/A) upstream of the CDH13 gene at 16q23.3. The initial associations with HYP and DBP were confirmed in two other European population-based cohorts: KORA S4 (Germans) and HYPEST (Estonians). The associations between rs11646213 and three BP traits were replicated in combined analyses (dominant model: DBP, P = 5.55 × 10–5, effect –1.40 mmHg; SBP, P = 0.007, effect –1.56 mmHg; HYP, P = 5.30 × 10−8, OR = 0.67). Carriers of the minor allele A had a decreased risk of hypertension. A non-significant trend for association was also detected with severe family based hypertension in the BRIGHT sample (British). The novel susceptibility locus, CDH13, encodes for an adhesion glycoprotein T-cadherin, a regulator of vascular wall remodeling and angiogenesis. Its function is compatible with the BP biology and may improve the understanding of the pathogenesis of hypertension.

Targeting 160 Candidate Genes for Blood Pressure Regulation with a Genome-Wide Genotyping Array

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<2.15×10−5), the strength of some detected associations was close to this level: rs10889553 (LEPR) and systolic BP (SBP) (P = 4.5×10−5) as well as rs10954174 (LEP) and diastolic BP (DBP) (P = 5.20×10−5). 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.

Combined Effects of the Variants FSHB −211G>T and FSHR 2039A>G on Male Reproductive Parameters

CONTEXT A polymorphism in the FSHB promoter (-211G>T, rs10835638) was shown to influence male serum FSH levels, whereas a polymorphism in the FSH receptor gene (FSHR; 2039A>G, rs6166) was previously shown to be associated with FSH levels in women only. OBJECTIVE The objective of the study was to analyze the effects of both FSHB -211G>T and FSHR 2039A>G on male reproductive parameters. DESIGN AND SETTING A total of 1213 German men attending an infertility clinic were genotyped by TaqMan assay. PATIENTS Patients included male partners in infertile couples without known causes for male infertility. MAIN OUTCOME MEASURES An association analysis of single and combined single-nucleotide polymorphism genotypes with clinical parameters was performed. RESULTS The FSHB -211G>T T-allele showed significant dosage effects for FSH (-0.51 U/liter per T-allele), LH (0.28 U/liter), and bitesticular volume (-3.2 ml). Statistical significance was enhanced severalfold after a meta-analysis comprising 3017 men. TT carriers were significantly more prevalent among men with lower sperm counts. The FSHR 2039A>G G-allele exhibited nonsignificant trends for associations with higher FSH and reduced testicular volumes. However, in the combined model, FSHR 2039A>G significantly modulated the more dominant effect of FSHB -211G>T on serum FSH and testicular volume among the T-allele carriers. CONCLUSIONS By analyzing both single-nucleotide polymorphisms for the first time, we convincingly show that indeed FSHR 2039A>G has an effect also in males. In the proposed model of the combined effects, FSHB -211G>T acts strongly on male reproductive parameters, whereas the FSHR 2039A>G effects were approximately 2-3 times smaller. Clinically this is of importance because oligozoospermic patients carrying unfavorable variants affecting FSH action may benefit from FSH treatment.

Study in 1790 Baltic men: FSHR Asn680Ser polymorphism affects total testes volume

Follicle‐stimulating hormone receptor (FSHR) contains two common linked polymorphisms, Thr307Ala (rs6165) and Asn680Ser (rs6166), shown to modulate ovarian function in women. The effect on male fertility and reproductive parameters has been inconclusive. We studied FSHR Asn680Ser polymorphism in a large study group (n = 1790) from the Baltic countries. The population‐based Baltic male cohort (Estonians, Latvians, Lithuanians; n = 1052) and Estonian oligo‐/azoospermic (sperm concentration <20 × 106/mL) idiopathic infertile patients (n = 738) were genotyped for the FSHR Asn680Ser using PCR‐RFLP. Genetic associations were tested using linear regression under additive model and results were combined in meta‐analysis. No statistical difference was detected in allelic distribution of the FSHR Asn680Ser between the Baltic cohort and Estonian male infertility group. A consistent significant association was detected between the FSHR Ser680 allele and lower total testes volume in both, the Baltic cohort (p = 0.010, effect = −1.16 mL) and Estonian idiopathic infertility group (p = 0.007, effect = −1.77 mL). In meta‐analysis, the statistical significance was enhanced (p = 0.000066, effect = −1.40 mL). Meta‐analysis supported further associations with moderate effect between the FSHR Ser680 variant and higher serum FSH (p = 0.072), lower Inhibin B (p = 0.037) and total testosterone (p = 0.034). No statistically significant associations were identified with serum LH and estradiol, and sperm parameters. In conclusion, the study in 1790 Baltic men shows statistically highly significant association of the FSHR Asn680Ser with total testes volume and supportive association with serum reproductive hormone levels indicative to the functional effect of the alternative FSHR variants on male reproductive physiology.

Beyond Genome-Wide Association Studies: New Strategies for Identifying Genetic Determinants of Hypertension

Genetic linkage and association methods have long been the most important tools for gene identification in humans. These approaches can either be hypothesis-based (i.e., candidate-gene studies) or hypothesis-free (i.e., genome-wide studies). The first part of this review offers an overview of the latest successes in gene finding for blood pressure (BP) and essential hypertension using these DNA sequence–based discovery techniques. We further emphasize the importance of post–genome-wide association study (post-GWAS) analysis, which aims to prioritize genetic variants for functional follow-up. Whole-genome next-generation sequencing will eventually be necessary to provide a more comprehensive picture of all DNA variants affecting BP and hypertension. The second part of this review discusses promising novel approaches that move beyond the DNA sequence and aim to discover BP genes that are differentially regulated by epigenetic mechanisms, including microRNAs, histone modification, and methylation.

Extensive shift in placental transcriptome profile in preeclampsia and placental origin of adverse pregnancy outcomes

One in five pregnant women suffer from gestational complications, prevalently driven by placental malfunction. Using RNASeq, we analyzed differential placental gene expression in cases of normal gestation, late-onset preeclampsia (LO-PE), gestational diabetes (GD) and pregnancies ending with the birth of small-for-gestational-age (SGA) or large-for-gestational-age (LGA) newborns (n = 8/group). In all groups, the highest expression was detected for small noncoding RNAs and genes specifically implicated in placental function and hormonal regulation. The transcriptome of LO-PE placentas was clearly distinct, showing statistically significant (after FDR) expressional disturbances for hundreds of genes. Taqman RT-qPCR validation of 45 genes in an extended sample (n = 24/group) provided concordant results. A limited number of transcription factors including LRF, SP1 and AP2 were identified as possible drivers of these changes. Notable differences were detected in differential expression signatures of LO-PE subtypes defined by the presence or absence of intrauterine growth restriction (IUGR). LO-PE with IUGR showed higher correlation with SGA and LO-PE without IUGR with LGA placentas. Whereas changes in placental transcriptome in SGA, LGA and GD cases were less prominent, the overall profiles of expressional disturbances overlapped among pregnancy complications providing support to shared placental responses. The dataset represent a rich catalogue for potential biomarkers and therapeutic targets.

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.

Age-dependent association of the polymorphisms in the mitochondria-shaping gene, OPA1, with blood pressure and hypertension in Korean population.

BACKGROUND Essential hypertension is associated with mitochondrial dysfunction. Because mitochondrial dynamics; mitochondrial morphological changes are closely linked with various mitochondrial functions, we aimed to examine whether the genetic variation of the mitochondria-shaping genes influenced the susceptibility to blood pressure (BP) and hypertension. METHODS The quantitative BP trait analysis and hypertension case-control analysis for the total 52 single-nucleotide polymorphisms (SNPs) in the five major mitochondria-shaping genes were performed in the Korean Association Resource (KARE) study cohort (8,512 subjects). RESULTS In the total subjects of the KARE study cohort, there were no statistically significant associations of the SNPs in the five mitochondria-shaping genes with BP or hypertension after adjusting for multiple tests. However, the age group analysis in the 40s, 50s, and 60s age subgroups revealed that 15 SNPs out of 26 SNPs genotyped in the OPA1 gene were significantly associated with BP and/or hypertension in the 60s age subgroup and their association P values satisfied the Bonferroni-corrected significance level (P < 0.00625). Noticeably, nine SNPs were consistently associated with all the three traits; systolic BP (SBP), diastolic BP (DBP), and hypertension. In silico lookup of the associated SNPs in the Southern German population did not reveal associations with BP traits. CONCLUSIONS Our results indicate that genetic variation of the mitochondrial fusion-regulating gene, OPA1, might be associated with BP and hypertension in an age-dependent and population-specific manner in the Korean study cohort, and suggest that altered mitochondrial dynamics, especially involved in the mitochondrial fusion event, may play an important role in the pathogenesis of hypertension.

Hypervariable intronic region in NCX1 is enriched in short insertion-deletion polymorphisms and showed association with cardiovascular traits

BackgroundConserved non-coding regions (CNR) have been shown to harbor gene expression regulatory elements. Genetic variations in these regions may potentially contribute to complex disease susceptibility.MethodsWe targeted CNRs of cardiovascular disease (CVD) candidate gene, Na(+)-Ca(2+) exchanger (NCX1) with polymorphism screening among CVD patients (n = 46) using DHPLC technology. The flanking region (348 bp) of the 14 bp indel in intron 2 was further genotyped by DGGE assay in two Eastern-European CVD samples: essential hypertension (HYPEST; 470 cases, 652 controls) and coronary artery disease, CAD (CADCZ; 257 cases, controls 413). Genotype-phenotype associations were tested by regression analysis implemented in PLINK. Alignments of primate sequences were performed by ClustalW2.ResultsNine of the identified NCX1 variants were either singletons or targeted by commercial platforms. The 14 bp intronic indel (rs11274804) was represented with substantial frequency in HYPEST (6.82%) and CADCZ (14.58%). Genotyping in Eastern-Europeans (n = 1792) revealed hypervariable nature of this locus, represented by seven alternative alleles. The alignments of human-chimpanzee-macaque sequences showed that the major human variant (allele frequency 90.45%) was actually a human-specific deletion compared to other primates. In humans, this deletion was surrounded by other short (5-43 bp) deletion variants and a duplication (40 bp) polymorphism possessing overlapping breakpoints. This indicates a potential indel hotspot, triggered by the initial deletion in human lineage. An association was detected between the carrier status of 14 bp indel ancestral allele and CAD (P = 0.0016, OR = 2.02; Bonferroni significance level alpha = 0.0045), but not with hypertension. The risk for the CAD development was even higher among the patients additionally diagnosed with metabolic syndrome (P = 0.0014, OR = 2.34). Consistent with the effect on metabolic processes, suggestive evidence for the association with heart rate, serum triglyceride and LDL levels was detected (P = 0.04).ConclusionsCompared to SNPs targeted by large number of locus-specific and genome-wide assays, considerably less attention has been paid to short indel variants in the human genome. The data of genome dynamics, mutation rate and population genetics of short indels, as well as their impact on gene expressional profile and human disease susceptibility is limited. The characterization of NCX1 intronic hypervariable non-coding region enriched in human-specific indel variants contributes to this gap of knowledge.

RNA sequencing of chorionic villi from recurrent pregnancy loss patients reveals impaired function of basic nuclear and cellular machinery

Recurrent pregnancy loss (RPL) concerns ~3% of couples aiming at childbirth. In the current study, transcriptomes and miRNomes of 1st trimester placental chorionic villi were analysed for 2 RPL cases (≥6 miscarriages) and normal, but electively terminated pregnancies (ETP; n = 8). Sequencing was performed on Illumina HiSeq 2000 platform. Differential expression analyses detected 51 (27%) transcripts with increased and 138 (73%) with decreased expression in RPL compared to ETP (DESeq: FDR P < 0.1 and DESeq2: <0.05). RPL samples had substantially decreased transcript levels of histones, regulatory RNAs and genes involved in telomere, spliceosome, ribosomal, mitochondrial and intra-cellular signalling functions. Downregulated expression of HIST1H1B and HIST1H4A (Wilcoxon test, fc≤0.372, P≤9.37 × 10−4) was validated in an extended sample by quantitative PCR (RPL, n = 14; ETP, n = 24). Several upregulated genes are linked to placental function and pregnancy complications: ATF4, C3, PHLDA2, GPX4, ICAM1, SLC16A2. Analysis of the miRNA-Seq dataset identified no large disturbances in RPL samples. Notably, nearly 2/3 of differentially expressed genes have binding sites for E2F transcription factors, coordinating mammalian endocycle and placental development. For a conceptus destined to miscarriage, the E2F TF-family represents a potential key coordinator in reprogramming the placental genome towards gradually stopping the maintenance of basic nuclear and cellular functions.