Decision letter: Patient-specific genomics and cross-species functional analysis implicate LRP2 in hypoplastic left heart syndrome

Field: Medicine (Health Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to parents the proband's iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts. Introduction Hypoplastic left heart syndrome (HLHS) is a congenital heart disease (CHD) characterized by underdevelopment of the left ventricle, mitral and aortic valves, and aortic arch. Variable phenotypic manifestations and familial inheritance patterns, together with the numerous studies linking it to a diverse array of genes, suggest that HLHS is genetically heterogeneous and may have significant environmental contributors (Elliott et al., 2003; Iascone et al., 2012; Theis et al., 2015a; Theis et al., 2015b). In this scenario, synergistic combinations of filtering and validating approaches are necessary to prioritize candidate genes and gene variants that may affect cardiogenic pathways throughout the dynamic process of human heart development. Although the cellular mechanisms for HLHS remain poorly characterized, a recent study reported generation of the first animal model of HLHS. Based on a digenic mechanism, mice deficient for HDAC-associated protein-encoding Sap130 and protocadherin-coding Pcdha9 exhibited left ventricular (LV) hypoplasia that was likely due – at least in part – to defective cardiomyocyte proliferation and differentiation, and increased cell death (Liu et al., 2017). Similarly in humans, Gaber et al., 2013 provide evidence that HLHS-LV samples have more DNA damage and senescence with cell cycle arrest, and fewer cardiac progenitors and myocytes than controls. These observations suggest that impaired cardiomyocyte proliferation could be a mechanism contributing to HLHS pathogenesis, although pathogenic genes controlling this process in humans remain to be identified and validated. Therefore, new synergistic experimental approaches are needed to functionally evaluate gene candidates potentially involved in defective cardiogenesis to serve as a platform for probing the postulated oligogenic basis of CHDs, such as HLHS (Hinton et al., 2007; McBride et al., 2005). Over the last decade, induced pluripotent stem cells (iPSCs) have provided a revolutionary experimental tool to reveal aspects of the cellular manifestations associated with disease pathogenesis (Matsa et al., 2016; Mercola et al., 2013; Moretti et al., 2013). Progress in next-generation sequencing technology allows rapid whole-genome DNA and RNA sequencing, thereby providing access to high-resolution and personalized genetic information. However, the interpretation of patient-specific sequence variants is often challenged by uncertainty in establishing a pathogenic link between biologically relevant variant(s) and a complex disease (Cooper and Shendure, 2011). Testing numerous potential human disease-susceptibility genes in a mammalian in vivo model has been challenging because of high costs and low throughput. Drosophila with its genetic tools has emerged as the low cost, high-throughput model of choice for human candidate disease gene testing, including neurological and cardiac diseases (Fink et al., 2009; Ocorr et al., 2014; Şentürk and Bellen, 2018; Schroeder et al., 2019; Vissers et al., 2020; van der Harst et al., 2016). Drosophila has been established as an efficient model system to identify key genes and mechanisms critical for heart development and function that served as prototypes for vertebrate/mammalian studies, including in zebrafish and mice, due to high degree of conservation of genetic pathways and reduced genetic complexity (Bodmer and Frasch, 2010), e.g. the first cardiogenic transcription factor Nkx2-5/tinman, discovered in Drosophila (Azpiazu and Frasch, 1993; Bodmer, 1993), marked the beginning of a molecular-genetic understanding of cardiac specification (Bodmer, 1995; Cripps and Olson, 2002; Benson et al., 1999; Cordes and Srivastava, 2009; Qian and Srivastava, 2013; Kathiriya et al., 2015; Fahed et al., 2013). For this study, we combined whole-genome sequencing (WGS), iPSC technology and model system validation with a family-based approach to identify and characterize novel HLHS-associated candidate genes and postulate potential mechanisms involved. This approach led us to identify LRP2 as a major regulator of cardiac cardiomyocyte proliferation of hiPSCs and heart development and maturation in both Drosophila and zebrafish. Consistent with our model system findings, burden analysis revealed that rare and predicted deleterious missense LRP2 variants were enriched in HLHS patients as compared to healthy controls. Finally, we found evidence consistent with LRP2 regulating cardiac proliferation and differentiation by potentially modulating growth-associated WNT, SHH, and TP53 pathways. Importantly, our integrated multidisciplinary high-throughput approach establishes a scalable and synergistic gene discovery platform to investigate potential oligogenic participants in genetically complex forms of human heart diseases. Results Transcriptome and cell cycle activity are altered in HLHS patient-derived iPSCs and CMs This study analyzed a family comprised of unrelated parents and their three offspring ('5H' family; Figure 1A). The male proband (II.3) was diagnosed with non-syndromic HLHS by physical examination and echocardiography, which demonstrated aortic and mitral valve atresia, virtual absence of the left ventricular cavity, and severe aortic arch hypoplasia. He was born prematurely at 29 weeks gestation and underwent staged surgical palliation at 2 and 11 months of age. Conversion to a fenestrated Fontan circulation at 3 years of age failed owing to systolic and diastolic heart failure, necessitating early take-down. The patient subsequently died of multi-organ system failure. Echocardiography revealed structurally and functionally normal hearts in the proband's mother (I.2), father (I.1) and siblings (II.1 and II.2). Maternal history is notable for four miscarriages. Figure 1 with 2 supplements see all Download asset Open asset Family-based iPSC characterization for HLHS. (A) Pedigree of family 5H: proband with HLHS (black symbol), relatives without CHD (white symbols), miscarriages (gray diamonds). (B) Schematic for family-based iPSC production and characterization. (C) Whole-genome RNA sequencing identified 1401 concordantly DETs between proband and parents. (D) KEGG pathway analysis shows enrichment of DETs in TP53 pathway. (E) Heatmap of p53 signaling pathway-associated genes in probands vs parents. (F) Schematic describing EdU-incorporation assay in hiPSC-CMs. 5000 cells/well were plated in 384 well plates. After 48 hr EdU was added to the media and left incorporate for 24 hr. Cells were then fixed and stained (G) Graph representing quantification of EdU+ cardiomyocytes in HLHS 5H family-derived iPSC-CMs. ***p<0.001 one-way ANOVA. (H) Representative images of iPSC-CMs derived from mother (Top) and proband (Bottom), stained for EdU, ACTN1 and DAPI. Scale bar: 50 µm. (I) Quantification of EdU-incorporation assay in 5H proband iPSC-CM upon KD of TP53 or CDKN1A. ****p<0.0001, one-way ANOVA. (J) Representative images of 5H proband iPSC-CM stained for EdU and ACTN1 upon KD of TP53 or CDKN1A at day 28. Scale bar: 50 µm. Patient-derived iPSCs are a valuable tool to investigate heart defects, such as those observed in HLHS (Theis et al., 2015a; Hrstka et al., 2017). In this study, iPSCs from the mother (I.2), father (I.1) and HLHS proband (II.3) were generated (Takahashi and Yamanaka, 2006) to investigate differences in transcriptional profiles potentially associated with HLHS. Cells from the proband-parent trio were differentiated to day 25 (d25), using a cardiogenic differentiation protocol and processed for subsequent RNA sequencing (Figure 1B). In this in vitro cellular context, bioinformatic analysis revealed 5104 differentially expressed transcripts (DETs) in d25 differentiated samples between proband vs. mother/father (Supplementary file 1, Benjamini-corrected p<0.001). We found that 1,401 DETs were concordantly differentially expressed between proband and both parents (Figure 1C, Figure 1—figure supplement 1, Supplementary files 1, 2). Consistent with previous observations in HLHS fetuses (Gaber et al., 2013), KEGG analysis revealed TP53 pathway enrichment (Figure 1D), including cell cycle inhibition (Figure 1E and Figure 1—figure supplement 1), consistent with cell proliferation being affected in proband cells. To begin exploring this hypothesis, we measured cell cycle activity in proband and parent hiPSC-derived cardiomyocytes (hiPSC-CMs) using an EdU-incorporation assay (Figure 1F). Indeed, proband hiPSC-CMs exhibited reduced percentage of EdU-positive cells as compared to parents (Figure 1G,H). To further evaluate whether a potentially reduced proliferative activity is a more general phenotypic hallmark of HLHS cells, we evaluated the proliferative status of two additional HLHS family trios that were available to us from the HLHS cohort at Mayo Clinic ('75H', '151H'). Consistent with our findings with 5H family-trio cells (Figure 1G,H), the proband cells of families 75H and 151H also exhibited significant reduction of proliferative activity as compared to the parents using the EdU-incorporation assay (Figure 1—figure supplement 2). Given the upregulation of potent cell cycle inhibitors TP53 or CDKN1A in 5H proband cells (Figure 1E), we tested whether impaired proliferation could involve the observed elevated TP53 and/or CDKN1A mRNA levels. Indeed, siRNA-mediated knockdown (KD) of TP53 and CDKN1A in proband hiPSC-CMs significantly increased EdU incorporation as compared to siControl (Figure 1I,J). These findings are consistent with a CM proliferation defect observed in both HLHS fetuses (Gaber et al., 2013) and a HLHS mouse model (Liu et al., 2017). Family-based WGS, variant filtering, and transcriptional profiling identified 10 candidates Array comparative genome hybridization ruled out a chromosomal deletion or duplication in the proband. WGS was carried out on genomic DNA samples from all five family members, based on 101 base paired-end reads that passed quality control standards; 92% of the reads mapped to the genome. After marking and filtering out duplicate reads, over 99% of the hg19 human reference genome had coverage. The average depth across the genome was 36X and an average of 91% of the gene body regions (exons, introns, and 5' and 3' untranslated regions) demonstrated a minimal read depth of 20 reads. WGS was performed to identify potentially pathogenic coding or regulatory single nucleotide variants (SNVs) or insertion/deletions (INDELs). First, we ruled out pathogenic variants within 42 genes comprising a CHD genetic testing panel (Invitae, San Francisco, CA). To identify novel HLHS candidate genes, WGS of the family quintet was filtered for rare de novo, recessive and loss-of-function variants with predicted impact on protein structure or expression, yielding 114 variants in 61 genes (Figure 2, Figure 2—figure supplement 1,, Supplementary file 3). We next prioritized genes most likely to drive downstream pathways of dysregulated cardiogenesis in the HLHS proband by cross-referencing these candidate genes with 3,816 DETs identified in undifferentiated iPSC at d0 (Supplementary file 4) and 5,104 DETs identified at d25 differentiated cell lineages (Supplementary file 1). Ten genes harboring compound heterozygous (7), hemizygous (2), or homozygous (1) recessive variants (Table 1), absent in the unaffected siblings, were found to be differentially expressed within the HLHS proband's iPSCs at d0 and d25: HSPG2, APOB, LRP2, PRTG, SLC9A1, SDHD, JPT1, ELF4, HS6ST2, and SIK1 (Figure 2A). qPCR confirmed reduced expression of these genes in proband in d25 iPSC-CM, compared to the parental cells (Figure 2—figure supplement 2). In order to explore if and how these genes could affect cardiac differentiation and/or function, alone or in combination, we employed an integrated gene discovery platform using multiple genetic model systems (see below). We consider this approach an efficient first pass evaluation of the potential roles of these genes in the heart; roles that need to be further substantiated by validation of patient-specific variants, also in a combinatorial fashion, based on the oligogenic hypothesis of CHDs. Figure 2 with 4 supplements see all Download asset Open asset Whole-genome and RNA sequencing identify HLHS candidate genes. (A) An iterative, family-based variant filtering approach based on rarity, functional impact, and mode of inheritance and RNA sequencing data were used to filter for transcriptional differences yielding 10 candidate genes. Candidate genes were further tested in hiPSC-CM and in vivo model. (B) Human candidate genes and corresponding Drosophila ortholog as determined by DIOPT score (*confidence score: number of databases reporting orthology). Listed are heart phenotypes upon gene candidate KD. (C,D) Example of fly hearts heterozygous for LRP2/mgl show increased end-diastolic diameters (EDD, measured at green line in D). Wilcoxon rank sum test: ***p<0.001. (E) Graph representing EdU-incorporation assay results of candidate gene KD in hiPSC-CM. KD of APOB (red bar) or LRP2 (green bar) reduced EdU incorporation. **p<0.01 one-way ANOVA. (F) Representative images of hiPSC-CMs stained for EdU, ACTN1 and DAPI. Scale bar: 50 µm. (G) qPCR results of TP53 and CDKN1A in hiPSC-CM upon KD of APOB or LRP2. *p<0.05 one-way ANOVA. Table 1 Recessive Variants Identified in 10 Candidate Genes. GeneMode of inheritanceFunctional impactTranscript variantProtein variantInheritanceGenotype in brother (II.1)Genotype in sister (II.2)gnomAD* MAF (%)dbSNP IDHSPG2Cmpd Hetmissensec.2074G > A; c.2077G > Ap.V692M; p.V693MMaternalWTWT0.288143669458missensec.326G > Ap.R109QPaternalHetHet0773796176promoterc.-227C > APaternalWTWT1.392566166086SLC9A1Cmpd Hetpromoterc.-906T > CPaternalHetHet1.227114101904promoterc.-947T > GMaternalWTWT27.17511588974promoterc.-1085A > GPaternalHetHet0.841116299278ENCODE TFBSc.-1138C > TPaternalHetHet0.9375089536promoterc.-1311G > APaternalHetHet0.9377414471APOBCmpd Hetmissensec.13441G > Ap.A4481TMaternalWTHet2.4751801695missensec.751G > Ap.A251TPaternalHetWT0.07161741625LRP2Cmpd Hetmissensec.9613A > Gp.N3205DMaternalWTWT0.40735734447missensec.170C > Tp.A57VPaternalWTWT0.032115350461SDHDCmpd Hetpromoterc.-815G > C; c.129+547C > GMaternalHetWT0.573117661257ENCODE TFBSc.-205G > A; c.66C > Tp.A22APaternalWTWT0.24161734353missensec.34G > A; c.-173C > Tp.G12SMaternalHetWT0.72934677591PRTGCmpd HetmicroRNA Binding Sitec.*3501T > GPaternalHetWT0.73977181316microRNA Binding Sitec.*2678A > GMaternalWTHet0.019756136447HN1Cmpd HetENCODE TFBSc.56+617C > T; c.-903C > T; c.-178+617C > T; c.-590C > TMaternalHetWT3.764117213586promoterc.-1748A > C; c.-719A > C; c.-486A > CPaternalWTHet0.81673995795SIK1Hom Recmissensec.2087C > Tp.P696LMaternal and PaternalWTHet1256991707ELF4X-Linkedmissensec.1144G > Ap.V382IMaternalWTHet0.025148953158HS6ST2X-Linkedmissensec.948–40041G > A; c.1046G > Ap.R349QMaternalWTHet0.146201239951 Cmpd Het, compound heterozygous; Het, heterozygous; Hom Rec, homozygous recessive; MAF, minor allele frequency; WT, wild- type. *At study initiation the ESP database was used to set the 3% allele frequency filter. Updated frequencies are shown based on the newer gnomAD database curation which would now eliminate SLC9A1 and HN1 as candidate genes. Knockdown of candidate gene orthologs in Drosophila heart In order to determine whether these variants occurred within genes that could be important for cardiac differentiation in vivo, we took advantage of our established Drosophila heart development model and functional analysis tools (Figure 2—figure supplement 3; Ocorr et al., 2014). We hypothesized that genes critical for the Drosophila heart have conserved roles, also in humans, as previously observed (Bodmer, 1995; Cripps and Olson, 2002; Qian et al., 2011). Predicted by DIOPT database (Hu et al., 2011) to have orthologs in Drosophila (Figure 2B), we analyzed nine genes using heart-specific RNAi-KD. By in vivo heart structure and function analysis (Fink et al., 2009; Ocorr et al., 2014), we found that KD of LRP2 (mgl) and JPT1 (CG1943) caused dilated heart phenotypes, while KD of APOB (apolpp), a circulating lipoprotein ligand, and again LRP2 (mgl), resulted in arrhythmias (Figure 2B–D, Figure 2—figure supplement 3; Videos 1–3), suggesting developmental defects of cardiac structure and function. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Dissected adult fly heart showing rhythmic beating pattern. Representative heart movies of dissected adult females showing arrhythmic beating pattern in APOB-RNAi (Video 2) and LRP2-RNAi (Video 3) compared to control hearts (Video 1). All movies are imaged at 140 frames/sec. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg APOB-RNAi causes arrhythmia in dissected adult fly hearts. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg LRP2-RNAi causes arrhythmia in dissected adult fly hearts. Since HLHS is likely oligogenic (Blue et al., 2017; Gelb and Chung, 2014), functional requirements for some genes involved in HLHS might only become apparent in combination with variants in other cardiac-relevant genes. To test this, we examined the nine candidates in the heterozygous background for which in humans is to to a of manifestations (Elliott et al., 2003; Hrstka et al., 2017; et al., 2014). In this in vivo context, heart-specific KD of two out of nine genes, involved in et al., and exhibited a (Figure 2—figure supplement These findings that our bioinformatic candidate gene identified conserved candidates as cardiac further validation is necessary to begin to link in a to HLHS. LRP2 and APOB proliferation in human iPSC-derived cardiomyocytes proliferation of left ventricular cardiomyocytes is as a phenotypic hallmark of HLHS (Liu et al., 2017; Gaber et al., 2013) (see also Figure and Figure 1—figure supplement suggesting that cell cycle may be an important contributing we whether siRNA-mediated KD of the prioritized 10 candidate genes from the 5H family trio (Figure proliferation of normal hiPSC-CM et al., 2017). two of the genes cardiac in Drosophila (Figure LRP2 and APOB, also caused a marked reduction of EdU+ hiPSC-CMs and cell (Figure and Figure 2—figure supplement we also observed an upregulation of cell cycle inhibitors and genes (Figure 2—figure supplement including TP53 and CDKN1A (Figure as well as a of cell cycle genes (Figure 2—figure supplement Collectively, these data identify LRP2 and APOB as of cell cycle and in however, further validation is necessary to link to contributing to the developmental cardiac in HLHS variant analysis in HLHS cohort enrichment in LRP2 In order to explore disease of candidate genes functionally in both we whether the frequency of rare and predicted-damaging variants in LRP2 and APOB would be in a cohort of HLHS compared to control HLHS patients had a 3 in the frequency of rare, predicted-damaging LRP2 missense variants compared to healthy (Figure Supplementary file the patients carried a LRP2 variant (Figure three the predicted-damaging variant with the 5H proband (Figure Supplementary file of the HLHS the family of while all of the had rare, predicted-damaging missense variants in LRP2. However, all variants were required to be rare in all To eliminate the potentially of a was in a significant for rare, predicted-damaging missense variants However, of the predicted-damaging on rare LRP2 variants revealed significant enrichment in most in missense and variants and Supplementary file allele and of variants within functional protein or transcription factor was between and controls. Figure 3 with 1 supplement see all Download asset Open asset of LRP2 as potential HLHS candidate (A) analysis of LRP2 variants shows significant enrichment for in HLHS patients compared to control Variants are found throughout LRP2 (B) Table the HLHS families LRP2 (C,D) qPCR of LRP2 in 5H family (C) and in family (D) showing LRP2 in parent and proband compared to the one-way one-way ANOVA. (E) in zebrafish at were significantly reduced in the (F) cardiomyocyte number was also reduced in to a than in hearts were by expression in the background at hearts were and compared to controls. background cardiomyocyte used for cardiomyocytes during development in and the in Scale In a next we to determine whether LRP2 might be affected in probands with rare, predicted-damaging variants in LRP2 coding We LRP2 transcripts in patient-derived iPSCs of the 5H family as well as both harboring heterozygous variants with a score 24 (Figure from of the parents. Interestingly, qPCR results that LRP2 mRNA were in the probands of both as well as in the parent the variant compared to parent (Figure We the protein coding variants (see Table are associated with reduced RNA in patient-derived which we may be due to reduced of the variant LRP2 or altered LRP2 function could to This the that these LRP2 variants and may be a genetic in the 5H and However, are likely other contributing the of the LRP2 variants, CHD in parents. LRP2 loss-of-function results in a hypoplastic ventricular In order to evaluate the of LRP2 during heart development in a we a as well as LRP2 in zebrafish and evaluated the on heart and function at body was for and at compared to (Figure supplement from with reduced function a hypoplastic with CM number (Figure and in ventricular in (Figure supplement also ventricular and caused in both and (Figure supplement Collectively, our data suggest that LRP2 a crucial during heart development by regulating cardiomyocyte generation most in the ventricular regulatory of gene candidates In order to how the candidate genes testing in our validation APOB, HS6ST2, HSPG2, JPT1, LRP2, might affect signaling we a gene these five genes and their first genetic and (Figure Supplementary file in to TP53 pathway (Figure Figure and Figure 2—figure supplement all five genes were to and signaling both key of cardiac differentiation and proliferation et al., 2016; and Figure Interestingly, analysis of the proband cells is consistent with this the regulator of was while of signaling and et al., 2013), were compared to parental cells. However, genetic studies in our model systems are required in order to a link between these two pathways and LRP2 in CM proliferation. Figure 4 Download asset Open asset for SHH, and LRP2 in HLHS. (A) gene HLHS candidate genes with heart development. – genes with cardiac phenotypes in – other candidate genes with Drosophila – in proband iPSC-CMs vs. parents. – in proband iPSCs vs. parents. qPCR for a (B) and for a pathway-associated gene (C) upon LRP2 KD in hiPSC-CM. *p<0.05 (D) Quantification of incorporation assay in hiPSC-CM upon LRP2 KD in combination with a ****p<0.0001, one-way ANOVA. (E) Representative images of hiPSC-CM stained for EdU and Scale 50 µm. (F) Quantification of EdU-incorporation assay in hiPSC-CM upon LRP2 KD in combination with a ****p<0.0001, one-way ANOVA. (G) Representative images of hiPSC-CM stained for EdU and Scale 50 µm. a first we examined whether LRP2 could and/or genes, using healthy control hiPSC-CM. Indeed, KD of LRP2 led to reduced and increased RNA (Figure although were we used the et al., 2006) and et al., in the of LRP2 We found that LRP2 KD significantly reduced both and proliferation in hiPSC-CMs (Figure suggesting that LRP2 is required for both and CM proliferation. are required to a link between LRP2 and signaling in heart and differentiation. Discussion multidisciplinary disease gene discovery platform the patient-specific molecular-genetic of HLHS pathogenesis our (1) to provide to families and to novel approaches to or the an important first toward these our integrated multidisciplinary approach is to identify variants and gene from WGS of family that are relevant for