HiPS-Endothelial Cells Acquire Cardiac Endothelial Phenotype in Co-culture With hiPS-Cardiomyocytes

Cell-cell interactions are crucial for organ development and function. In the heart, endothelial cells engage in bidirectional communication with cardiomyocytes regulating cardiac development and growth. We aimed to elucidate the organotypic development of cardiac endothelial cells and cardiomyocyte and endothelial cell crosstalk using human induced pluripotent stem cells (hiPSC). Single-cell RNA sequencing was performed with hiPSC-derived cardiomyocytes (hiPS-CMs) and endothelial cells (hiPS-ECs) in mono- and co-culture. The presence of hiPS-CMs led to increased expression of transcripts related to vascular development and maturation, cardiac development, as well as cardiac endothelial cell and endocardium-specific genes in hiPS-ECs. Interestingly, co-culture induced the expression of cardiomyocyte myofibrillar genes and MYL7 and MYL4 protein expression was detected in hiPS-ECs. Major regulators of BMP- and Notch-signaling pathways were induced in both cell types in co-culture. These results reflect the findings from animal studies and extend them to human endothelial cells, demonstrating the importance of EC-CM interactions during development.Peer reviewe

BMP4:n vaikutus rintasyövän kasvuun ja metastasointiin – näkökulmia transkriptionaaliseen säätelyyn

Rintasyöpä on maailmanlaajuisesti naisten yleisin syöpä. Luun morfogeneettiset proteiinit (bone morphogenetic protein, BMP) ovat signalointimolekyylejä, jotka ovat usein syövässä poikkeavalla tavalla säädeltyjä. BMP4:n on aiemmin näytetty hidastavan rintasyöpäsolujen kasvua ja joissakin tapauksissa samalla lisäävän niiden migraatiokykyä. Nämä tutkimukset on kuitenkin tehty standardimallisessa 2D kasvatuksessa. Tämän tutkimuksen tavoitteena oli tutkia BMP4:n vaikutusta rintasyöpäsoluihin 3D- ja in vivo -malleissa, sekä lisäksi tutkia BMP4-reitin kohdegeenejä ja signaalinsäätelyä. Usean eri rintasyöpäsolulinjan soluja kasvatettiin sekä synteettisessä PEG geelissä että biologisesta lähteestä saadussa Matrigeelissä. BMP4 hidasti solujen kasvua molemmissa materiaaleissa. Kasvun laskua tutkittiin lähemmin Matrigeelissä ja efektin todettiin johtuvan osittain p21 induktiosta. Lisäksi MDA-MB-231 rintasyöpäsolut Matrigeelissä muodostivat BMP4:n vaikutuksesta isoja haarautuvia rakenteita, jotka viittaavat lisääntyneeseen migraatioon/invaasioon. Tämä reaktio oli matriksin metalloproteinaaseista riippuvainen. BMP4:n aiheuttamaa migraatio/invaasioefektiä tutkittiin tarkemmin käyttämällä hiirimallia ja seuraamalla BMP4:n kykyä vaikuttaa metastaasien muodostukseen. Hiirten sydämiin injektoitiin MDA-MB-231 soluja ja niitä käsiteltiin BMP4:llä tai vehikkelikontrollilla. BMP4-käsitellyissä hiirissä oli jonkin verran enemmän luumetastaaseja, mutta vähemmän lisämunuaismetastaaseja kuin vehikkeliryhmässä. Metastaaseista tutkittiin BMP signaloinnin aktivoitumista, kasvua, epiteeli-mesenkymaalitransitiota sekä verisuonia ja syöpään liittyviä fibroblasteja (cancer-associated fibroblasts). Eroa ryhmien välillä ei kuitenkaan ollut. Mielenkiintoista oli että molemmissa ryhmissä löytyi osteoklastimarkkerin värjäytymistä syöpäsolujen joukosta. BMP4:n signaloinnin tutkimusta varten MDA-MB-231 ja T-47D rintasyöpäsoluja käsiteltiin BMP4:llä tai vehikkelillä ja analysoitiin geenien ilmentymiseroja (RNA-seq) ja genomissa olevia säätelyalueita (DNaasi-seq). RNA-seq data osoitti solulinjojen olevan keskenään hyvin erilaisia, vaikka myös yhteisiä BMP4 kohdegeenejä löytyi. Rikastumisanalyysi paljasti, että MDA-MB-231 soluissa, jotka reagoivat BMP4:ään lisääntyneellä migraatiolla, liikkumiseen liittyvät geenit olivat rikastuneet. Vastaavasti T-47D soluissa, joissa tapahtuu kasvun hidastuminen BMP4:n vaikutuksesta, kehitykseen ja signalointiin liittyvät geenit olivat rikastuneet. Samanlaiset tulokset saatiin kun analysoitiin BMP4:n aiheuttamien avoimen kromatiinin alueiden rikastumista. Lisäksi avoimen kromatiinin analysoinnin avulla valittiin kolme transkriptiotekijää, MBD2, CBFB ja HIF1A, joiden validoitiin funktionaalisten siRNA-kokeiden perusteella olevan BMP4-reitin alavirran säätelijöitä. Näistä MBD2 toimi säätelyn aktivaattorina molemmissa solulinjoissa, CBFB T-47D soluissa ja HIF1A toimi repressorina MDA-MB-231 soluissa. Yhteenvetona BMP4:llä on kasvua vähentäviä ja migraatiota lisääviä vaikutuksia sekä 2D että 3D kasvatuksissa, mutta lisätutkimuksia tarvitaan selventämään BMP4:n osuutta metastaasien muodostuksessa, erityisesti luumetastaaseissa. BMP4:n aiheuttamat muutokset heijastuvat geenien ilmentymiseen ja kromatiinin aukeamiseen. Lisäksi muutoksista riippuen eri transkriptiotekijät vaikuttavat säätelevän BMP4:n kohdegeenejä.Breast cancer is the most common cancer in women worldwide. The bone morphogenetic proteins (BMPs) are signaling molecules that are often aberrantly regulated in cancer. BMP4 has previously been shown to reduce the proliferation of breast cancer cells and in some cases increase their migration. However, these studies have been done using standard 2D culture. The aim of this study was to characterize the effect of BMP4 on breast cancer cells in 3D culture and using an in vivo model, as well as to study BMP4 target genes and signaling pathway regulation. Several different breast cancer cell lines were grown in both the synthetic PEG gel and biologically-derived Matrigel. BMP4 inhibited the proliferation of cells in both materials. The growth inhibition was examined more closely in Matrigel, showing that the effect was partly due to p21 induction. In addition, in response to BMP4 MDA-MB-231 breast cancer cells in Matrigel formed large, branching structures, indicative of increased migration/invasion. This reaction was dependent on matrix metalloproteinases. The migration/invasion effect promoted by BMP4 was examined in more detail by using a mouse model and following the effects of BMP4 on metastasis formation. The mice were injected intracardially with MDA-MB-231 cells and treated with BMP4 or vehicle control. The mice treated with BMP4 had slightly more bone metastases, but less adrenal gland metastases compared to the vehicle group. The activation of BMP signaling, epithelial-to-mesenchymal transition, as well as blood vessels and cancer-associated fibroblasts were studied from the metastases. However, there were no differences between the treatment groups. Interestingly, in both groups osteoclast marker staining was found among the cancer cells. In order to study BMP4 signaling, MDA-MB-231 and T-47D breast cancer cells were treated with BMP4 or vehicle and differences in gene expression (RNA-seq) and in regulatory regions of the genome (DNase-seq) were analyzed. RNA-seq data showed that the responses of the cell lines to BMP4 were different, although there were also common BMP4 target genes, which were also target genes in additional cell lines when tested with qPCR. Enrichment analysis revealed that in MDA-MB-231 cells, which react to BMP4 with increased migration, motility-related genes were enriched. Correspondingly, in T-47D cells, which respond with reduced proliferation, genes related to development and signaling were enriched. Similar results were obtained when analyzing enrichment of chromatin regions that were opened due to BMP4 treatment. Moreover, based on the open chromatin regions, three transcription factors (MBD2, CBFB and HIF1A) were chosen for functional analyses using siRNA and validated as BMP4 downstream regulators. Of these, MBD2 was mainly an activator in both cell lines, CBFB in T-47D cells and HIF1A acted as a repressor in MDA-MB-231 cells. Taken together, BMP4 inhibits proliferation and increases migration in both 2D and 3D culture, but more studies are needed to clarify the role of BMP4 in metastasis formation, particularly in bone metastases. The effects of BMP4 are reflected in gene expression and chromatin openness. Additionally, depending on the effects different transcription factors seem to regulate BMP4 target genes

BMP7 as a modulator of breast cancer growth and migration

Background and aims: Breast cancer is the most common cancer in women in Western countries and a global health concern affecting millions of people. Thus the search for causative agents and possible curatives continues to be important. Bone morphogenetic proteins (BMPs) are a group of growth factors that have been implicated in various cancers, including breast cancer. They were originally discovered due to their ability to induce bone formation, but are now recognized as multifunctional developmental regulators of cell proliferation, differentiation, apoptosis and motility. Dysregulation of BMP signaling pathways has been shown to contribute to cancer progression. The aim of this study was to examine the effect of BMP7 on breast cancer cells through proliferation, migration and invasion studies in five breast cancer cell lines. Methods: Breast cancer cells (MDA-MB-361, MDA-MB-231, HCC1954, SK-BR-3 and BT-474) were treated with recombinant human BMP7 (50 ng/ml) or vehicle control followed by functional assays. Effects on proliferation were measured by counting the cells and cell cycle analyses were performed using PI staining and flow cytometry. Migration and invasion were studied using the transwell assay. Finally, signaling cascade activation upon BMP7 stimulation was analyzed with Western blotting. Results: BMP7 treatment resulted in cell line-specific phenotypes. BMP7 decreased the growth of MDA-MB-361 and HCC1954 cells (on average 9% and 19%, respectively), increased the growth of MDA-MB-231 cells (128%) and had no effect on the two remaining cell lines. However, no differences in cell cycle were detected between BMP7- and vehicle-treated MDA-MB-361 and HCC1954 cells. BMP7 moderately increased migration in MDA-MB-361, SK-BR-3 and BT-474 (from 1.1-fold to 1.6-fold). A more dramatic increase (7.4-fold) was seen in MDA-MB-231 cells. Furthermore, invasion of BT-474 cells was examined but no changes were detected between BMP7- and vehicle-treated cells. Western blot showed that the canonical BMP signaling pathway through SMAD proteins was activated in MDA-MB-231, BT-474 and MDA-MB-361 cells, whereas in HCC1954 and SK-BR-3 cells the MAPK pathway was induced. Conclusions: BMP7 has diverse effects on the growth of breast cancer cells. BMP7 induces both proliferative and anti-proliferative effects, as well as having no influence on cell growth. Moderate increases are seen in migration upon BMP7 stimulation. In conclusion, BMP7 has an important and complex role in breast cancer, and its effects are dependent on cellular background

BMP4:n vaikutus rintasyövän kasvuun ja metastasointiin – näkökulmia transkriptionaaliseen säätelyyn

Rintasyöpä on maailmanlaajuisesti naisten yleisin syöpä. Luun morfogeneettiset proteiinit (bone morphogenetic protein, BMP) ovat signalointimolekyylejä, jotka ovat usein syövässä poikkeavalla tavalla säädeltyjä. BMP4:n on aiemmin näytetty hidastavan rintasyöpäsolujen kasvua ja joissakin tapauksissa samalla lisäävän niiden migraatiokykyä. Nämä tutkimukset on kuitenkin tehty standardimallisessa 2D kasvatuksessa. Tämän tutkimuksen tavoitteena oli tutkia BMP4:n vaikutusta rintasyöpäsoluihin 3D- ja in vivo -malleissa, sekä lisäksi tutkia BMP4-reitin kohdegeenejä ja signaalinsäätelyä. Usean eri rintasyöpäsolulinjan soluja kasvatettiin sekä synteettisessä PEG geelissä että biologisesta lähteestä saadussa Matrigeelissä. BMP4 hidasti solujen kasvua molemmissa materiaaleissa. Kasvun laskua tutkittiin lähemmin Matrigeelissä ja efektin todettiin johtuvan osittain p21 induktiosta. Lisäksi MDA-MB-231 rintasyöpäsolut Matrigeelissä muodostivat BMP4:n vaikutuksesta isoja haarautuvia rakenteita, jotka viittaavat lisääntyneeseen migraatioon/invaasioon. Tämä reaktio oli matriksin metalloproteinaaseista riippuvainen. BMP4:n aiheuttamaa migraatio/invaasioefektiä tutkittiin tarkemmin käyttämällä hiirimallia ja seuraamalla BMP4:n kykyä vaikuttaa metastaasien muodostukseen. Hiirten sydämiin injektoitiin MDA-MB-231 soluja ja niitä käsiteltiin BMP4:llä tai vehikkelikontrollilla. BMP4-käsitellyissä hiirissä oli jonkin verran enemmän luumetastaaseja, mutta vähemmän lisämunuaismetastaaseja kuin vehikkeliryhmässä. Metastaaseista tutkittiin BMP signaloinnin aktivoitumista, kasvua, epiteeli-mesenkymaalitransitiota sekä verisuonia ja syöpään liittyviä fibroblasteja (cancer-associated fibroblasts). Eroa ryhmien välillä ei kuitenkaan ollut. Mielenkiintoista oli että molemmissa ryhmissä löytyi osteoklastimarkkerin värjäytymistä syöpäsolujen joukosta. BMP4:n signaloinnin tutkimusta varten MDA-MB-231 ja T-47D rintasyöpäsoluja käsiteltiin BMP4:llä tai vehikkelillä ja analysoitiin geenien ilmentymiseroja (RNA-seq) ja genomissa olevia säätelyalueita (DNaasi-seq). RNA-seq data osoitti solulinjojen olevan keskenään hyvin erilaisia, vaikka myös yhteisiä BMP4 kohdegeenejä löytyi. Rikastumisanalyysi paljasti, että MDA-MB-231 soluissa, jotka reagoivat BMP4:ään lisääntyneellä migraatiolla, liikkumiseen liittyvät geenit olivat rikastuneet. Vastaavasti T-47D soluissa, joissa tapahtuu kasvun hidastuminen BMP4:n vaikutuksesta, kehitykseen ja signalointiin liittyvät geenit olivat rikastuneet. Samanlaiset tulokset saatiin kun analysoitiin BMP4:n aiheuttamien avoimen kromatiinin alueiden rikastumista. Lisäksi avoimen kromatiinin analysoinnin avulla valittiin kolme transkriptiotekijää, MBD2, CBFB ja HIF1A, joiden validoitiin funktionaalisten siRNA-kokeiden perusteella olevan BMP4-reitin alavirran säätelijöitä. Näistä MBD2 toimi säätelyn aktivaattorina molemmissa solulinjoissa, CBFB T-47D soluissa ja HIF1A toimi repressorina MDA-MB-231 soluissa. Yhteenvetona BMP4:llä on kasvua vähentäviä ja migraatiota lisääviä vaikutuksia sekä 2D että 3D kasvatuksissa, mutta lisätutkimuksia tarvitaan selventämään BMP4:n osuutta metastaasien muodostuksessa, erityisesti luumetastaaseissa. BMP4:n aiheuttamat muutokset heijastuvat geenien ilmentymiseen ja kromatiinin aukeamiseen. Lisäksi muutoksista riippuen eri transkriptiotekijät vaikuttavat säätelevän BMP4:n kohdegeenejä.Breast cancer is the most common cancer in women worldwide. The bone morphogenetic proteins (BMPs) are signaling molecules that are often aberrantly regulated in cancer. BMP4 has previously been shown to reduce the proliferation of breast cancer cells and in some cases increase their migration. However, these studies have been done using standard 2D culture. The aim of this study was to characterize the effect of BMP4 on breast cancer cells in 3D culture and using an in vivo model, as well as to study BMP4 target genes and signaling pathway regulation. Several different breast cancer cell lines were grown in both the synthetic PEG gel and biologically-derived Matrigel. BMP4 inhibited the proliferation of cells in both materials. The growth inhibition was examined more closely in Matrigel, showing that the effect was partly due to p21 induction. In addition, in response to BMP4 MDA-MB-231 breast cancer cells in Matrigel formed large, branching structures, indicative of increased migration/invasion. This reaction was dependent on matrix metalloproteinases. The migration/invasion effect promoted by BMP4 was examined in more detail by using a mouse model and following the effects of BMP4 on metastasis formation. The mice were injected intracardially with MDA-MB-231 cells and treated with BMP4 or vehicle control. The mice treated with BMP4 had slightly more bone metastases, but less adrenal gland metastases compared to the vehicle group. The activation of BMP signaling, epithelial-to-mesenchymal transition, as well as blood vessels and cancer-associated fibroblasts were studied from the metastases. However, there were no differences between the treatment groups. Interestingly, in both groups osteoclast marker staining was found among the cancer cells. In order to study BMP4 signaling, MDA-MB-231 and T-47D breast cancer cells were treated with BMP4 or vehicle and differences in gene expression (RNA-seq) and in regulatory regions of the genome (DNase-seq) were analyzed. RNA-seq data showed that the responses of the cell lines to BMP4 were different, although there were also common BMP4 target genes, which were also target genes in additional cell lines when tested with qPCR. Enrichment analysis revealed that in MDA-MB-231 cells, which react to BMP4 with increased migration, motility-related genes were enriched. Correspondingly, in T-47D cells, which respond with reduced proliferation, genes related to development and signaling were enriched. Similar results were obtained when analyzing enrichment of chromatin regions that were opened due to BMP4 treatment. Moreover, based on the open chromatin regions, three transcription factors (MBD2, CBFB and HIF1A) were chosen for functional analyses using siRNA and validated as BMP4 downstream regulators. Of these, MBD2 was mainly an activator in both cell lines, CBFB in T-47D cells and HIF1A acted as a repressor in MDA-MB-231 cells. Taken together, BMP4 inhibits proliferation and increases migration in both 2D and 3D culture, but more studies are needed to clarify the role of BMP4 in metastasis formation, particularly in bone metastases. The effects of BMP4 are reflected in gene expression and chromatin openness. Additionally, depending on the effects different transcription factors seem to regulate BMP4 target genes

BMP7 as a modulator of breast cancer growth and migration

Background and aims: Breast cancer is the most common cancer in women in Western countries and a global health concern affecting millions of people. Thus the search for causative agents and possible curatives continues to be important. Bone morphogenetic proteins (BMPs) are a group of growth factors that have been implicated in various cancers, including breast cancer. They were originally discovered due to their ability to induce bone formation, but are now recognized as multifunctional developmental regulators of cell proliferation, differentiation, apoptosis and motility. Dysregulation of BMP signaling pathways has been shown to contribute to cancer progression. The aim of this study was to examine the effect of BMP7 on breast cancer cells through proliferation, migration and invasion studies in five breast cancer cell lines. Methods: Breast cancer cells (MDA-MB-361, MDA-MB-231, HCC1954, SK-BR-3 and BT-474) were treated with recombinant human BMP7 (50 ng/ml) or vehicle control followed by functional assays. Effects on proliferation were measured by counting the cells and cell cycle analyses were performed using PI staining and flow cytometry. Migration and invasion were studied using the transwell assay. Finally, signaling cascade activation upon BMP7 stimulation was analyzed with Western blotting. Results: BMP7 treatment resulted in cell line-specific phenotypes. BMP7 decreased the growth of MDA-MB-361 and HCC1954 cells (on average 9% and 19%, respectively), increased the growth of MDA-MB-231 cells (128%) and had no effect on the two remaining cell lines. However, no differences in cell cycle were detected between BMP7- and vehicle-treated MDA-MB-361 and HCC1954 cells. BMP7 moderately increased migration in MDA-MB-361, SK-BR-3 and BT-474 (from 1.1-fold to 1.6-fold). A more dramatic increase (7.4-fold) was seen in MDA-MB-231 cells. Furthermore, invasion of BT-474 cells was examined but no changes were detected between BMP7- and vehicle-treated cells. Western blot showed that the canonical BMP signaling pathway through SMAD proteins was activated in MDA-MB-231, BT-474 and MDA-MB-361 cells, whereas in HCC1954 and SK-BR-3 cells the MAPK pathway was induced. Conclusions: BMP7 has diverse effects on the growth of breast cancer cells. BMP7 induces both proliferative and anti-proliferative effects, as well as having no influence on cell growth. Moderate increases are seen in migration upon BMP7 stimulation. In conclusion, BMP7 has an important and complex role in breast cancer, and its effects are dependent on cellular background

Flow-Induced Transcriptomic Remodeling of Endothelial Cells Derived From Human Induced Pluripotent Stem Cells

The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development.Peer reviewe

BMP4 inhibits the proliferation of breast cancer cells and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells in 3D environment

Background Bone morphogenetic protein 4 (BMP4) belongs to the transforming growth factor β (TGF-β) family of proteins. BMPs regulate cell proliferation, differentiation and motility, and have also been reported to be involved in cancer pathogenesis. We have previously shown that BMP4 reduces breast cancer cell proliferation through G1 cell cycle arrest and simultaneously induces migration in a subset of these cell lines. Here we examined the effects of BMP4 in a more physiological environment, in a 3D culture system. Methods We used two different 3D culture systems; Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel. AlamarBlue reagent was used for cell proliferation measurements and immunofluorescence was used to determine cell polarity. Expression of cell cycle regulators was examined by Western blot and matrix metalloproteinase (MMP) expression by qRT-PCR. Results The MCF-10A normal breast epithelial cells formed round acini with correct apicobasal localization of α6 integrin in Matrigel whereas irregular structures were seen in PEG gel. The two 3D matrices also supported dissimilar morphology for the breast cancer cells. In PEG gel, BMP4 inhibited the growth of MCF-10A and the three breast cancer cell lines examined, thus closely resembling the 2D culture conditions, but in Matrigel, no growth inhibition was observed in MDA-MB-231 and MDA-MB-361 cells. Furthermore, BMP4 induced the expression of the cell cycle inhibitor p21 both in 2D and 3D culture, thereby partly explaining the growth arrest. Interestingly, MDA-MB-231 cells formed large branching, stellate structures in response to BMP4 treatment in Matrigel, suggestive of increased cell migration or invasion. This effect was reversed by Batimastat, a broad-spectrum MMP inhibitor, and subsequent analyses showed BMP4 to induce the expression of MMP3 and MMP14, that are thus likely to be responsible for the stellate phenotype. Conclusions Taken together, our results show that Matrigel provides a more physiological environment for breast epithelial cells than PEG gel. Moreover, BMP4 partly recapitulates in 3D culture the growth suppressive abilities previously seen in 2D culture and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells.BioMed Central open acces

Genome-wide association studies highlight novel risk loci for septal defects and left-sided congenital heart defects

Background Congenital heart defects (CHD) are structural defects of the heart affecting approximately 1% of newborns. They exhibit low penetrance and non-Mendelian patterns of inheritance as varied and complex traits. While genetic factors are known to play an important role in the development of CHD, the specific genetics remain unknown for the majority of patients. To elucidate the underlying genetic risk, we performed a genome wide association study (GWAS) of CHDs in general and specific CHD subgroups using the FinnGen Release 10 (R10) (N > 393,000), followed by functional fine-mapping through eQTL and co-localization analyses using the GTEx database. Results We discovered three genome-wide significant loci associated with general CHD. Two of them were located in chromosome 17: 17q21.32 (rs2316327, intronic: LRRC37A2, Odds ratio (OR) [95% Confidence Interval (CI)] = 1.17[1.12–1.23], p = 1.5 × 10–9) and 17q25.3 (rs1293973611, nearest: BAHCC1, OR[95%CI] = 4.48[2.80–7.17], p = 7.0 × 10–10), respectively, and in addition to general CHD, the rs1293973611 locus was associated with the septal defect subtype. The third locus was in band 1p21.2 (rs35046143, nearest: PALMD, OR[95%CI] = 1.15[1.09–1.21], p = 7.1 × 10–9), and it was associated with general CHD and left-sided lesions. In the subgroup analysis, two additional loci were associated with septal defects (rs75230966 and rs6824295), and one with left-sided lesions (rs1305393195). In the eQTL analysis the variants rs2316327 (general CHD), and rs75230966 (septal defects) both located in 17q21.32 (with a LD r2 of 0.41) were both predicted to significantly associate with the expression of WNT9B in the atrial appendage tissue category. This effect was further confirmed by co-localization analysis, which also implicated WNT3 expression in the atrial appendage. A meta-analysis of general CHD together with the UK Biobank (combined N = 881,678) provided a different genome-wide significant locus in LRRC37A2; rs16941382 (OR[95%CI] = 1.15[1.11–1.20], p = 1.5 × 10–9) which is in significant LD with rs2316327. Conclusions Our results of general CHD and different CHD subcategories identified a complex risk locus on chromosome 17 near BAHCC1 and LRRC37A2, interacting with the genes WNT9B, WNT3 and MYL4, may constitute potential novel CHD risk associated loci, warranting future experimental tests to determine their role.peerReviewe