Investigation of rare and low-frequency variants using high-throughput sequencing with pooled DNA samples

High-throughput sequencing using pooled DNA samples can facilitate genome-wide studies on rare and low-frequency variants in a large population. Some major questions concerning the pooling sequencing strategy are whether rare and low-frequency variants can be detected reliably, and whether estimated minor allele frequencies (MAFs) can represent the actual values obtained from individually genotyped samples. In this study, we evaluated MAF estimates using three variant detection tools with two sets of pooled whole exome sequencing (WES) and one set of pooled whole genome sequencing (WGS) data. Both GATK and Freebayes displayed high sensitivity, specificity and accuracy when detecting rare or low-frequency variants. For the WGS study, 56% of the low-frequency variants in Illumina array have identical MAFs and 26% have one allele difference between sequencing and individual genotyping data. The MAF estimates from WGS correlated well (r = 0.94) with those from Illumina arrays. The MAFs from the pooled WES data also showed high concordance (r = 0.88) with those from the individual genotyping data. In conclusion, the MAFs estimated from pooled DNA sequencing data reflect the MAFs in individually genotyped samples well. The pooling strategy can thus be a rapid and cost-effective approach for the initial screening in large-scale association studies.Peer reviewe

Gene expression profiling of pre-eclamptic placentae by RNA sequencing

Pre-eclampsia is a common and complex pregnancy disorder that often involves impaired placental development. In order to identify altered gene expression in pre-eclamptic placenta, we sequenced placental transcriptomes of nine pre-eclamptic and nine healthy pregnant women in pools of three. The differential gene expression was tested both by including all the pools in the analysis and by excluding some of the pools based on phenotypic characteristics. From these analyses, we identified altogether 53 differently expressed genes, a subset of which was validated by qPCR in 20 cases and 19 controls. Furthermore, we conducted pathway and functional analyses which revealed disturbed vascular function and immunological balance in pre-eclamptic placenta. Some of the genes identified in our study have been reported by numerous microarray studies (BHLHE40, FSTL3, HK2, HTRA4, LEP, PVRL4, SASH1, SIGLEC6), but many have been implicated in only few studies or have not previously been linked to pre-eclampsia (ARMS2, BTNL9, CCSAP, DIO2, FER1L4, HPSE, LOC100129345, LYN, MYO7B, NCMAP, NDRG1, NRIP1, PLIN2, SBSPON, SERPINB9, SH3BP5, TET3, TPBG, ZNF175). Several of the molecules produced by these genes may have a role in the pathogenesis of pre-eclampsia, and some could qualify as biomarkers for prediction or detection of this pregnancy complication.Peer reviewe

Exome sequencing in pooled DNA samples to identify maternal pre-eclampsia risk variants

Pre-eclampsia is a common pregnancy disorder that is a major cause for maternal and perinatal mortality and morbidity. Variants predisposing to pre-eclampsia might be under negative evolutionary selection that is likely to keep their population frequencies low. We exome sequenced samples from a hundred Finnish pre-eclamptic women in pools of ten to screen for low-frequency, large-effect risk variants for pre-eclampsia. After filtering and additional genotyping steps, we selected 28 low-frequency missense, nonsense and splice site variants that were enriched in the pre-eclampsia pools compared to reference data, and genotyped the variants in 1353 pre-eclamptic and 699 non-pre-eclamptic women to test the association of them with pre-eclampsia and quantitative traits relevant for the disease. Genotypes from the SISu project (n = 6118 exome sequenced Finnish samples) were included in the binary trait association analysis as a population reference to increase statistical power. In these analyses, none of the variants tested reached genome-wide significance. In conclusion, the genetic risk for pre-eclampsia is likely complex even in a population isolate like Finland, and larger sample sizes will be necessary to detect risk variants.Peer reviewe

Analysis of Complement C3 Gene Reveals Susceptibility to Severe Preeclampsia

Preeclampsia (PE) is a common vascular disease of pregnancy with genetic predisposition. Dysregulation of the complement system has been implicated, but molecular mechanisms are incompletely understood. In this study, we determined the potential linkage of severe PE to the most central complement gene, C3. Three cohorts of Finnish patients and controls were recruited for a genetic case-control study. Participants were genotyped using Sequenom genotyping and Sanger sequencing. Initially, we studied 259 Finnish patients with severe PE and 426 controls from the Southern Finland PE and the Finnish population-based PE cohorts. We used a custom-made single nucleotide polymorphism (SNP) genotyping assay consisting of 98 SNPs in 18 genes that encode components of the complement system. Following the primary screening, C3 was selected as the candidate gene and consequently Sanger sequenced. Fourteen SNPs from C3 were also genotyped by a Sequenom panel in 960 patients with severe PE and 705 controls, including already sequenced individuals. Three of the 43 SNPs observed within C3 were associated with severe PE: rs2287845 (p = 0.038, OR = 1.158), rs366510 (p = 0.039, OR = 1.158), and rs2287848 (p = 0.041, OR = 1.155). We also discovered 16 SNP haplotypes with extreme linkage disequilibrium in the middle of the gene with a protective (p = 0.044, OR = 0.628) or a predisposing (p = 0.011, OR = 2.110) effect to severe PE depending on the allele combination. Genetic variants associated with PE are located in key domains of C3 and could thereby influence the function of C3. This is, as far as we are aware, the first candidate gene in the complement system with an association to a clinically relevant PE subphenotype, severe PE. The result highlights a potential role for the complement system in the pathogenesis of PE and may help in defining prognostic and therapeutic subgroups of preeclamptic women

Genetic risk of pre-eclampsia : maternal and fetal studies in a population isolate

Pre-eclampsia is a complex vascular pregnancy disorder characterised by systemic endothelial dysfunction and often impaired placental development. The course of the disease is unpredictable and severe forms of the disease may threaten the lives of both the mother and her child. Indeed, pre-eclampsia is a major cause for maternal and perinatal mortality, especially in countries with limited access to maternal and neonatal health care. The risk of pre-eclampsia involves a genetic component conveyed by both the maternal and fetal genomes. Based on the largest genome-wide association study on maternal pre-eclampsia, the risk of the disease is influenced by many of the genetic loci previously implicated in hypertension. However, the hypertension risk variants do not fully explain the genetic risk of pre-eclampsia and variants involved in other physiological processes, such as placental development, are also likely to play a role. As pre-eclampsia reduces reproductive success, any predisposing large-impact variants are assumed to be pruned out to low population frequencies by negative evolutionary selection. Deleterious low-frequency variation, on the other hand, is enriched in population isolates, where evolutionary selection has not had enough time to effectively prune out the harmful variation. Due to the consequent boost in statistical power to detect genetic associations, studies in population isolates, such as the Finnish population, offer a feasible option for exploring especially low-frequency pathogenic variation. The aim of this thesis was to identify genetic risk factors of pre-eclampsia in the Finnish population isolate. The thesis includes two studies on a protective candidate gene HMOX1 that is crucial for vascular and placental function and has previously been linked to cardiometabolic diseases. In the other two studies, we performed genome- and exome-wide scans in families and sporadic cases of pre-eclampsia, respectively. The main sources for the samples in the thesis were the Finnish Genetics of Pre-eclampsia Consortium (FINNPEC) case-control cohort and the pre-eclampsia family cohort predominantly originating from a north-eastern population isolate. Initial findings of the screening studies were also evaluated in samples from two large population-based studies, Sequencing Initiative Suomi (SISu) and FinnGen. In the HMOX1 studies, we found that a functional promoter microsatellite was associated with both maternal and fetal pre-eclampsia. The long maternal repeat was associated with the late-onset, less severe phenotype whereas the fetal association was observed especially for the early-onset and severe forms of the disease. In accordance with these findings, we confirmed the previously reported impact of the microsatellite length on the heme oxygenase 1 (HO-1) protein levels, but here for the first time during pregnancy. The long maternal repeat was associated with reduced gestational serum HO-1 while the long fetal repeat showed an association with decreased HO-1 levels in placenta. These results suggest that HMOX1 is associated with pre-eclampsia through both maternal and fetal genomes, likely acting through separate mechanisms. In the exome-wide screen, we searched for pre-eclampsia-enriched low-frequency variants based on whole-exome sequencing data from a hundred pre-eclamptic women and aimed to validate the identified candidates by testing their association with pre-eclampsia in a larger set of case-control samples (1344 cases and 6817 controls). Four variants were nominally associated with pre-eclampsia, but none of them reached statistical significance after correcting for multiple testing. We conclude that larger sample sizes will likely be needed to reveal large-impact risk variants of sporadic pre-eclampsia, even in a population isolate like Finland. In the family study of pre-eclampsia, we performed linkage analysis in 15 extended families, both for the maternal and fetal phenotypes, using genome-wide imputed SNP markers and parametric linkage analysis. We identified 15 maternal and 27 fetal loci that segregated with the disease. In order to evaluate if there was an overlap between the risk loci in the families and the general population, we tested whether the suggestive case-control association signals from the maternal meta-analysis of ~90 000 samples and fetal analysis of ~1700 samples would be enriched in the linkage regions. Evidence of such enrichment was, however, not observed in our study, implying that the risk loci identified might be largely specific to the families. Finally, we searched for linkage region candidate variants in 21 exome sequenced pre-eclamptic women from 6 families and genotyped the identified variants in the whole family cohort. Four previously unknown or rare missense variants in the linkage region genes COL6A3, DGKB, EEPD1, and PITPNM1 were found to segregate with pre-eclampsia either fully or partially. As each of the candidates was found in only one of the families, additional support from independent genetic or functional studies will be required to establish their connection with pre-eclampsia. In conclusion, the studies of the thesis shed light on genetic susceptibility of pre-eclampsia by identifying novel risk variants and loci for both the maternal and fetal phenotypes. We also demonstrate the benefits of utilising the family and population isolate based designs, especially in the search for rare risk variants of complex reproductive diseases such as pre-eclampsia.Pre-eklampsia on raskauden aikana ilmenevä monitekijäinen verisuonitauti. Tautiin liittyy äidin endoteelin toimintahäiriön lisäksi usein heikentynyttä istukankehitystä. Pre-eklampsia on yksi yleisimmistä äitiys- ja lapsikuolleisuuden syistä etenkin maissa, joissa äitiyshuollon resurssit ovat puutteelliset. Sekä äidin että lapsen geneettiset tekijät vaikuttavat alttiuteen sairastua pre-eklampsiaan, mutta suurinta osaa riskitekijöistä ei vielä tunneta. Tutkimuksen tavoitteena oli löytää pre-eklampsian geneettisiä riskitekijöitä suomalaisessa väestössä, johon on rikastunut harvinaisia haitallisia tekijöitä. Kaksi väitöskirjan osatöistä keskittyi verisuonten ja istukan toiminnalle tärkeään suojaavaan geeniin HMOX1, joka on aiemmin liitetty kardiometabolisiin sairauksiin. Väitöskirja sisältää myös kaksi genomin- tai eksominlaajuista tutkimusta pre-eklampsiasuvuissa ja tapauksissa ilman tunnettua sukurasitetta. HMOX1-tutkimuksissa havaitsimme geenin säätelyalueen mikrosatelliittitoistojakson liittyvän pre-eklampsia-alttiuteen sekä äideillä että lapsilla. Äidin pitkä toistojakso liittyi myöhäiseen ja lievempään pre-eklampsiaan, kun taas lapsen toistojakso liittyi erityisesti varhaiseen ja vaikeaan pre-eklampsiaan. Pitkän toistojakson on aiemmin havaittu vähentävän HO-1-proteiinin määrää, ja vahvistimme tämän ensimmäistä kertaa raskauden aikaisissa näytteissä. Äidin pitkä toistojakso liittyi alentuneeseen HO-1-proteiinin määrään seerumissa raskauden aikana, kun taas lapsen pitkä toistojakso liittyi alentuneeseen HO-1-tasoon istukassa. Tulostemme perusteella geenin vaikutus pre-eklampsia-alttiuteen välittyy sekä äidin että sikiön perimän kautta, todennäköisesti erillisillä mekanismeilla. Eksominlaajuisessa tutkimuksessa etsimme pre-eklampsianäytteissä rikastuneita harvinaisia geneettisiä tekijöitä tutkimalla sadan pre-eklamptikon proteiinia koodaavat alueet. Jatkotutkimukseen valituista geneettisistä tekijöistä mikään ei riittävällä varmuudella liittynyt pre-eklampsiaan isommassa tapaus-verrokkiaineistossa. Pre-eklampsian harvinaisten geneettisten riskitekijöiden tunnistamiseen vaaditaan todennäköisesti suurempi määrä näytteitä jopa eristyneessä väestössä. Viidentoista pre-eklampsiaperheen aineistossa tunnistimme 15 äidin ja 27 lapsen kytkentäaluetta, jotka periytyivät pre-eklampsian kanssa. Emme havainneet tutkimuksessa viitteitä siitä, että alueet olisivat jaettuja sukujen ja muun väestön kesken. Etsimme kytkentäalueilta geneettisiä riskitekijöitä kuutta perhettä edustavista 21 eksomisekvensoidusta naisesta. Neljä aiemmin tuntematonta tai harvinaista proteiinin aminohapposekvenssiä muuttavaa varianttia kytkentäalueiden geeneissä COL6A3, DGKB, EEPD1, ja PITPNM1 periytyi perheissä joko osittain tai täydellisesti pre-eklampsian mukana. Kukin variantti havaittiin kuitenkin vain yhdessä perheessä, ja niiden varmentaminen pre-eklampsiaan liittyviksi tekijöiksi vaatii siksi tuekseen lisätutkimuksia. Tässä väitöskirjatyössä havaitsimme uusia pre-eklampsian geneettisiä riskitekijöitä ja -alueita sekä äidin että lapsen perimästä. Väitöskirjan tutkimukset tuovat siten lisätietoa pre-eklampsian geneettisestä alttiudesta

Prematura och fullgångna fenotyper i Finnish Genetics of Pre-eclampsia Consortium (FINNPEC) -kohorten

Introduktion: Preeklampsi är ett syndrom med en mångsidig fenotyp. Vi forskade om kliniska egenskaper av prematura och fullgångna graviditeter i FINNPEC (Finnish Genetics of Pre-eclampsia Consortium) -kohorten Material och metoder: Vi undersökte kliniska fenotyper hos 2541 kvinnor i FINNPEC. Angiogena markörer var tillgängliga i en delkohort. Jämförelser utfördes mellan fyra grupper: prematur preeklampsi, fullgången preeklampsi, prematur kontroll och fullgången kontroll. Resultat: Av 1460 kvinnor med preeklampsi födde 37,9 % prematurt och i kontrollgruppen bestående av 1081 kvinnor födde 6,8 % prematurt. Kroppsmasseindexet (BMI) före graviditeten var högre i prematur preeklampsi jämfört med prematurkontrollen och i fullgången preeklampsi jämfört med fullgångna kontrollen. Prevalensen av kronisk hypertoni ökade både i prematur preeklampsi- och kontrollgrupper jämfört med deras respektive fullgångna grupper. Mellan de fullgångna grupperna, drabbades kvinnor med preeklampsi oftare av kronisk hypertoni. Kvinnor i de prematura grupperna födde oftare barn med låg födelsevikt för åldern (SGA) och barnen hade lägre relativt födelsevikt än i de respektive fullgångna grupperna. Prematura kontroller födde oftare barn med låg födelsevikt för åldern jämfört med prematur preeklampsigruppen och samma gällde kvinnor i de fullgångna grupperna. Markörprofilen under graviditeten var mera antiangiogen i preeklampsigrupperna. Slutsatser: Det uppkom signifikanta skillnader i kliniska egenskaper och angiogena markörer mellan de fyra grupperna. Prematur preeklampsi- och kontrollgrupperna var märkbart liknande, vilket återspeglar en möjlig fenotypisk kontinuitet mellan dessa. (216 ord

Preeclampsia does not share common risk alleles in 9p21 with coronary artery disease and type 2 diabetes

Introduction: Preeclampsia is a common and partially genetic pregnancy complication characterized by hypertension and proteinuria. Association with cardiovascular disease and type 2 diabetes has been reported in 9p21 by several genome-wide association studies. It has been hypothesized that cardiometabolic diseases may share common etiology with preeclampsia. Materials and methods: We tested association with the 9p21 region to preeclampsia in the Finnish population by genotyping 23 tagging single nucleotide polymorphisms (SNPs) in 15 extended preeclampsia families and in a nationwide cohort consisting of 281 cases and 349 matched controls. Replication was conducted in additional datasets. Results: Four SNPs (rs7044859, rs496892, rs564398 and rs7865618) showed nominal association (p ≤ 0.024 uncorrected) with preeclampsia in the case-control cohort. To increase power, we genotyped two SNPs in additional 388 cases and 341 controls from the Finnish Genetics of Preeclampsia Consortium (FINNPEC) cohort. Partial replication was also attempted in a UK cohort (237 cases and 199 controls) and in 74 preeclamptic families from Australia/New Zealand. We were unable to replicate the initial association in the extended Finnish dataset or in the two international cohorts. Conclusions: Our study did not find evidence for the involvement of the 9p21 region in the risk of preeclampsia

An RGS2 3'UTR Polymorphism is Associated with Preeclampsia in Overweight Women

Background Preeclampsia is a common and heterogeneous vascular syndrome of pregnancy. Its genetic risk profile is yet unknown and may vary between individuals and populations. The rs4606 3' UTR polymorphism of the Regulator of G-protein signaling 2 gene (RGS2) in the mother has been implicated in preeclampsia as well as in the development of chronic hypertension after preeclampsia. The RGS2 protein acts as an inhibitor of physiological vasoconstrictive pathways, and a low RGS2 level is associated with hypertension and obesity, two conditions that predispose to preeclampsia. We genotyped the rs4606 polymorphism in 1339 preeclamptic patients and in 697 controls from the Finnish Genetics of Preeclampsia Consortium (FINNPEC) cohort to study the association of the variant with preeclampsia. Results No association between rs4606 and preeclampsia was detected in the analysis including all women. However, the polymorphism was associated with preeclampsia in a subgroup of overweight women (body mass index ≥ 25 kg/m2, and < 30 kg/m2) (dominant model; odds ratio, 1.64; 95% confidence interval, 1.10-2.42). Conclusions Our results suggest that RGS2 might be involved in the pathogenesis of preeclampsia particularly in overweight women and contribute to their increased risk for hypertension and other types of cardiovascular disease later in life