Background: Heterotaxy (Htx) syndrome comprises a class of congenital disorders resulting from malformations in left-right body patterning. Approximately 90% of patients with heterotaxy have serious congenital heart diseases; as a result, the survival rate and outcomes of Htx patients are not satisfactory. However, the underlying etiology and mechanisms in the majority of Htx cases remain unknown. The aim of this study was to investigate the function of rare copy number variants (CNVs) in the pathogenesis of Htx. Methods: We collected 63 sporadic Htx patients with congenital heart defects and identified rare CNVs using an Affymetrix CytoScan HD microarray and real-time polymerase chain reaction. Potential candidate genes associated with the rare CNVs were selected by referring to previous literature related to left-right development. The expression patterns and function of candidate genes were further analyzed by whole mount in situ hybridization, morpholino knockdown, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated mutation, and over-expressing methods with zebrafish models. Results: Nineteen rare CNVs were identified for the first time in patients with Htx. These CNVs include 5 heterozygous genic deletions, 4 internal genic duplications, and 10 complete duplications of at least one gene. Further analyses of the 19 rare CNVs identified six novel potential candidate genes (NUMB, PACRG, TCTN2, DANH10, RNF115,and TTC40)linkedto left-right patterning. These candidate genes exhibited early expression patterns in zebrafish embryos. Functional testing revealed that downregulation and over-expression of five candidate genes (numb, pacrg, tctn2, dnah10,and rnf115)in zebrafish resulted in disruption of cardiac looping and abnormal expression of lefty2 or pitx2, molecular markers of left-right patterning. Conclusions: Our findings show that Htx with congenital heart defects in some sporadic patients may be attributed to rare CNVs. Furthermore, DNAH10 and RNF115 are Htx candidate genes involved in left-right patterning which have not previously been reported in either humans or animals. Our results also advance understanding of the genetic components of Htx. Keywords: Copy number variants, Congenital heart defects, Heterotaxy, Zebrafish, Left-right * Correspondence: firstname.lastname@example.org; email@example.com Chunjie Liu and Ruixue Cao contributed equally to this work. Scientific Research Center, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Department of Pediatric Cardiology, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Genome Medicine (2018) 10:40 Page 2 of 13 Background heavy chain 10 (dnah10), and ring-finger protein 115 Heterotaxy (Htx) syndrome is a serious congenital malfor- (rnf115) in zebrafish resulted in disruption of both mation with high mortality and morbidity characterized heart looping and expression of lefty2 or pitx2.Toour by the failure to establish normal left-right (LR) body knowledge, this study is the first to identify DNAH10 asymmetry. Patients often present with abnormal arrange- and RNF115 as novel Htx candidate genes. These two ment of the thoraco-abdominal organs, including ectopia genes have not been previously implicated in LR patterning of the heart, lungs, spleen, or liver . The survival rate in either humans or animals; numb, pacrg,and tctn2 and outcomes of patients with Htx are unsatisfactory, as have been linked to LR development in animals but are approximately 90% of cases are associated with complex previously unreported in patients with Htx. congenital heart diseases, including malposition of the great arteries, presence of a single right ventricle, total Methods anomalous pulmonary venous drainage, and double-outlet Patient ascertainment and study populations right ventricle [2, 3]. Our study recruited patients with Htx in Xinhua Hospital Researchers have made significant progress in enhancing and Shanghai Children’s Medical Center (SCMC) whose our understanding of the molecular and cellular mecha- diagnoses were confirmed by echocardiography, cardiac nisms that determine laterality during early embryogenesis. catheterization examinations, computed tomography, In the primitive node, asymmetry signaling is activated by abdominal ultrasonography, and other operation recordings. leftward “nodal flow” created by the unidirectional rotation Patients exhibiting abnormal arrangement of the visceral of monocilia. The asymmetry signals are then transmitted to organs and complex congenital heart disease were included, the left lateral plate mesoderm, where they upregulate the while those with complete situs solitus or other syndromes expression of a series of left determinants, such as Nodal, were excluded. left-right determination factor 2 (Lefty2), and paired-like homeodomain 2 (Pitx2). Several signaling pathways are Affymetrix CytoScan HD microarray analysis involved in establishment of the LR axis, including Notch, Peripheral blood samples were obtained from each patient, Nodal, Hedgehog, Wnt, and transforming growth factor and DNA was extracted using the QIAamp DNA Blood beta (TGF-β)[3–5]. In humans, mutations in several genes Midi Kit (Qiagen, Duesseldorf, Germany) following the have been associated with Htx, including CFC1, NODAL, manufacturer’s instructions. The CNVs were detected ACVR2B, LEFTY2, GDF1, ZIC3, CRELD1,and NKX2.5 [6– by CytoScan HD microarray platform (Affymetrix, Santa 13]. However, the mutations reported in these genes can ex- Clara, CA, USA), which is a high-density chip that contains plain only 10–20% of Htx cases; the underlying cause in the 2,636,550 probes. In total, 59 samples passed initial quality majority of patients remains unknown [14–16]. control. Gains and losses were analyzed using Chromo- Copy number variants (CNVs) are DNA fragments some Analysis Suite (ChAS) software and the annotations whose copy number varies between individuals in a popu- of the Genome Reference Consortium (GRC) human lation due to duplication or deletion events. CNVs can reference genome version GRCh37 (hg19). The data range in size from 1 kilobase (kb) to several megabases were filtered, and only those regions larger than 50 kb (Mb). Numerous studies have demonstrated that a variety comprising at least 25 contiguous markers were considered. of diseases, especially syndrome-related diseases, are asso- Finally, we distinguished common CNVs from rare CNVs ciated with CNVs. Several researchers recently reported a by comparing the results with the known CNVs in the relationship between CNVs and Htx. Both Brueckner and Database of Genomic Variants (DGV, http://dgv.tcag.ca/) Mills identified several novel rare CNVs in congenital heart and Online Mendelian Inheritance in Man (OMIM, disease patients with abnormal LR patterning [15–17], http://omim.org). suggesting that CNVs may account for a proportion of patients. But the role of CNVs in the occurrence of Htx Quantitative real-time polymerase chain reaction in patients with complex congenital heart disease should validation be examined in greater detail. The selected segments, which are related with Htx, were In this study, we identified 19 rare CNVs in 63 patients verified by quantitative real-time polymerase chain reaction with Htx by genotyping their DNA using an Affymetrix (qPCR). The qPCR validation was performed according to CytoScan HD microarray. We further identified six poten- SYBR® Premix Ex TaqTM II protocol (Applied TaKaRa). tial candidate genes involved in several pathways reported We used 50 ng/μLofgenomic DNAina 20 μL reaction, to be related to LR development: ciliary proteome and func- consisting of 10 μLof2×SYBRPremixEx Taq,0.4 μLof tion, Notch signaling pathway, or ubiquitination (ubiquitin 50× ROX Reference Dye II, 0.3 μLofforward primer, ligase E3 family). Downregulation and over-expression of 0.3 μL of reverse primer, 8.0 μL of ddH O, and 1 μLof the Htx candidate genes numb, Parkin co-regulated gene DNA. GenomicDNAsextracted from healthypeoplewere (pacrg), tectonic family member 2 (tctn2), dynein axonemal mixed, serving as normal controls, and the house-keeping Liu et al. Genome Medicine (2018) 10:40 Page 3 of 13 gene GAPDH was used as the control in qPCR. The reac- gRNA into zebrafish embryos at the one-cell stage. The tions were performed in triplicate. gene mutation and knockout efficiency in F0 embryos was examinedbyPCR andsequencinganalysiswith the Whole-exome sequencing analysis and mutation following primers: detection rnf115 forward: 5′-GAGAAGCACTGGTTCCGTCA-3′. We performed whole-exome sequencing in patients with rnf115 reverse: 5′-AACATACCCCTCAACAGCGG-3′. Htx and also in 100 healthy control people. The DNA dnah10 forward: 5′-ATTCATCCAACGTGGAAAC was sequenced using the Illumina HiSeq 2500 platform CA-3′. at a commercial provider (Shanghai Biotechnology Co, dnah10 reverse: 5′-GTCAGGACCTCGGTTTATT Ltd., Shanghai, China). We defined functional mutations GTC-3′. to be nonsynonymous mutations, stop-gain mutations, The knockout efficiency in F0 embryos of dnah10 is stop-loss mutations, frameshift or non-frameshift deletions 84.6%; for rnf115 it is 75% (Additional file 2: Figure S2). or insertions, and splice site mutations. mRNA synthesis and injection Zebrafish husbandry Zebrafish total RNA was extracted from 1 to 2 days post Adult zebrafish (Danio rerio,ABlineandTg[cmlc2:EGFP] fertilization (dpf) wild-type embryos (AB strain) and line) were raised under standard laboratory conditions with then was retrotranscribed into coding DNA (cDNA) an automatic fish housing system (ESEN, Beijing, China) at TM with PrimeScript RT reagent (TaKaRa, Shiga, Japan) 28 °C. All zebrafish experiments were conducted at the according to the manufacturer’s instructions. The full-length Institute of Neuroscience, Chinese Academy of Sciences, coding sequence DNA of tctn2, pacrg, galnt11, numb,and according to standard protocols. Embryo stages were deter- rnf115 was amplified using specific primers with restriction mined according to their developmental morphology . enzyme digestion loci, which were next subcloned into the pCS2+ vector (Additional file 1: Table S2). Positive clones Morpholino oligo injection and target gene knockdown selected by DNA sequencing were applied to generate Morpholino oligos (MOs) designed against target genes the corresponding full-length mRNAs using a T7 or SP6 were purchased from Gene Tools (Philomath, OR, USA) mMessage mMachine kit (Ambion). For the over-expression and dissolved in nuclease-free water. According to Gene experiment, pacrg, tctn2, numb,and galnt11 mRNAs were Tools’ protocol, concentrations of MOs were checked by injected into one-cell-stage embryos and green fluorescent spectrophotometry (A265 in 0.1 N HCl). MOs were protein(GFP) wasusedasanegative control; fortherescue diluted to different working concentrations and 1 nL experiment, 6.25 pg rnf115 mRNA was injected into was pressure-injected into one-cell-stage embryos with a one-cell-stage embryos mixed with 2 pg rnf115 MO. Picospritzer II injector. The MOs for examining heart looping in morphants Whole mount in situ hybridization range from 2 ng to 8 ng in dosage: 8 ng pacrg MO, 8 ng The whole mount in situ hybridization (WMISH) with tctn2 MO, 8 ng numb MO, 8 ng dnah10 MO, 2 ng anti-digoxigenin probes was performed according to the rnf115 MO, 8 ng cfap46/ttc40 MO, and 8 ng galnt11 previously described protocol . The anti-digoxigenin MO per embryo. As negative controls, we injected 8 ng RNA probes (Roche) were synthesized with a length of of standard control MO. A summary of MO doses and 400–1300 necleotides, complementary to numb, pacrg, sequences is provided in Additional file 1: Table S1. The tctn2, dnah10, rnf115, pitx2, and lefty2, respectively knockdown efficiencies of splice blocking and translation (Additional file 1: Table S3). blocking MOs are illustrated in Additional file 2:FigureS1. The injected doses of MOs are from 2 ng to 8 ng for scoring of pitx2 and lefty2 expression in morphants: CRISPR/Cas9-mediated mutation of dnah10 and rnf115 in 8ng pacrg MO, 8 ng tctn2 MO, 8 ng numb MO, 8 ng zebrafish embryos dnah10 MO, 2 ng rnf115 MO, 8 ng cfap46/ttc40 MO, As previously reported, the clustered regularly inter- and 4 ng galnt11 MO per embryo. As negative controls, spaced short palindromic repeats (CRISPR)/CRISPR-asso- we injected 8 ng of standard control MO (Additional file 1: ciated protein 9 (Cas9) system was applied to introduce Table S1). dnah10 and rnf115 gene mutation in zebrafish embryos . The sequences of dnah10 guide RNA (gRNA) (5′-GGCTCAGTTCTATGCTTACT -3′)and rnf115 gRNA Statistical analysis (5′-GGACAGTCTTGACTCTGAG -3′)weredesignedto In all figures, statistical comparisons between groups were target the sequences of mature dnah10 and rnf115, analyzed by the chi-squared test (continuity corrected) or respectively. We co-injected 600 pg zCas9 messenger Fisher’s exact test. We defined P < 0.05 as statistically RNA (mRNA) and 100 pg dnah10 gRNA or 100 pg rnf115 significant with *P < 0.05, **P < 0.01, ***P < 0.001. Liu et al. Genome Medicine (2018) 10:40 Page 4 of 13 Results ubiquitin ligases were reported to play important roles in Clinical data the Nodal signaling pathway, cilia formation, and cilia A total of 63 Chinese children with sporadic Htx were assembly [22–24]. We finally found six potential candidate recruited. All of the patients exhibited abnormal arrange- genes in five CNV segments in five subjects: NUMB ment of the visceral organs and complex congenital heart [MIM: 603728], PACRG [MIM: 608427], TCTN2 [MIM: disease, not including complete situs solitus or other 613846], DANH10 [MIM: 605884], RNF115, and cilia and syndromes . Among the patients we recruited, no one flagella associated protein 46 [CFAP46/TTC40]. had central nervous system malformations, vertebral Verification by qPCR showed that these five rare defects, or genitourinary malformations. According to CNVs comprise four duplications and one deletion (Fig. 1 the patients’ medical history, there was no family history and Additional file 2: Figure S3). The qPCR results and of heterotaxy or other malformations. The patients’ ages clinical diagnosis of the five patients are summarized in ranged from 12 days to 113 months; 40 patients were male Table 3. (63.5%) and 23 were female (36.5%). The detailed cardiac To determine whether the patients identified as carrying and extracardiac clinical phenotypes are summarized in CNVs of the candidate genes had mutations in other Table 1. Pulmonary outflow obstruction was discovered known laterality-related genes (e.g., ZIC3, CFC1, NKX2.5, in 56 patients, complete or partial atrioventricular canal GDF1, NODAL, LEFTY1, LEFTY2, ACVR2B, DANH5, in 20 patients, and single atrium or single ventricle in DNAH11, DNAI1, FOXH1, CRELD1,and GALNT11), we 35 patients. Twenty-seven patients had malposed or screened the coding sequences of these genes using transposed great arteries, and 21 patients had double whole-exome sequencing analysis. Aside from a nonsynon- outlet of the right ventricle. ymous heterozygous mutation (c. 841A > G, p.Trp281Arg) in LEFTY1 (Additional file 2:FigureS4and Additional file 1: CNVs in patients with Htx and identification of candidate TableS6) in onepatient with a CNV of CFAP46,the results genes revealed no functional mutations in these laterality-related To identify the molecular causes of Htx, an Affymetrix genes. CytoScan HD microarray was used to identify possible pathogenic CNVs. A total of 59 samples passed initial Expression patterns of candidate genes in zebrafish quality control. Rare CNV segments were identified Zebrafish were used as a model organism to further based on the following criteria: (1) CNV > 50 kb in size; elucidate the roles of the candidate genes in regulating (2) > 25 markers in each segment; (3) present at < 1‰ organ laterality, as all six of the candidate genes have frequency or have < 50% overlap with published common orthologs in zebrafish. We examined the gene expression CNVs or not found in the DGV (http://dgv.tcag.ca/); (4) patterns at two developmental stages using WMISH with not identified in either a dataset with microarray results of digoxigenin-labeled RNA as probes: the 8–10 somite stage 216 normal Chinese individuals or another dataset of 720 (13–15 h post fertilization [hpf]; symmetry was first Chinese non-heterotaxy patients with developmental broken at Kupffer’s vesicle [KV]) and the primordium 5 delay/intellectual disability (DD/ID) (Additional file 1: stage (24 hpf; heart begins to beat). WMISH analysis of Table S4). Finally, we identified 19 rare CNVs in 14 the tctn2 gene was unsuccessful. The pacrg gene was patients with Htx (Table 2). The percentage of subjects expressed in the KV in the 8–10 somite stage, and was with rare CNVs was 23.7% (14 of 59 Htx subjects). The expressed in the pronephric duct and floor plate in the selected CNVs ranged in size from 57 to 1009 kb. These primordium 5 stage (Fig. 2a, b). In the 8–10 somite CNVs included 5 heterozygous genic deletions, 4 internal stage, numb, rnf115, dnah10,and cfap46 exhibited genic duplications, and 10 complete duplications of at nearly ubiquitous expression patterns (Fig. 2c, e, g, i). least one gene . In the primordium 5 stage, however, these four genes Among the 19 rare CNVs, none of the common exhibited more localized expression patterns: numb and known Htx-related genes, including ZIC3, CFC1, NKX2.5, rnf115 were both expressed in the brain (Fig. 2d, f); GDF1, NODAL, LEFTY1, LEFTY2, ACVR2B, DANH5, dnah10 was restricted to the notochord (Fig. 2h); DNAH11, DNAI1, FOXH1, CRELD1,or GALNT11,were cfap46 was expressed in the pronephric duct and floor identified. In order to correlate the phenotypes of the Htx plate (Fig. 2j). syndrome patients to specific pathologic genes, we first examined the function of the genes associated with the 19 Knockdown and mutation of the candidate genes rare CNV segments (Additional file 1: Table S5). Candi- disturbs cardiac looping date genes were identified based on the following criteria: Expression of the candidate genes in zebrafish was (1) participation in ciliary proteome and function; (2) rela- downregulated using MO knockdown to examine the tion to signaling pathways Notch, Nodal, Hedgehog, Wnt, effect on cardiac looping, which depends on normal LR and TGF-β; (3) member of ubiquitin ligase E3 family. E3 patterning. Three heart tube morphologies occur in Liu et al. Genome Medicine (2018) 10:40 Page 5 of 13 Table 1 Cardiac and extracardiac abnormalities in the patients Table 1 Cardiac and extracardiac abnormalities in the patients with Htx with Htx (Continued) Number of patients Number of patients (%) (%) Sex IVC Male 40 (63.5%) Interrupted IVC, hemiazygos vein continuation 1 (1.6%) Female 23 (36.5%) Interrupted IVC, azygos vein continuation 6 (9.5%) Cardiac position Relationship of IVC and descending aorta Levocardia 15 (23.8%) IVC right of spine and descending aorta left of 4 (6.3%) spine Dextrocardia 35 (55.6%) IVC left of spine and descending aorta right of 17 (27.0%) Mesocardia 13 (20.6%) spine Atrial arrangement IVC and descending aorta same side 32 (50.8%) Atrial situs inversus 20 (31.7%) IVC left of spine and descending aorta anterior 2 (3.2%) of spine Isomerism of right atrial appendages 33 (52.4%) IVC anterior of spine and descending aorta left 1 (1.6%) Isomerism of left atrial appendages 7 (11.1%) of spine Ventricular arrangement SVC superior vena cava, IVC inferior vena cava Ventricular situs solitus 14 (22.2%) Ventricular situs inversus 16 (25.4%) zebrafish: dextral loop (d-loop), sinistral loop (s-loop), or Single ventricle (morphologic right) 23 (36.5%) no loop (Fig. 3a). In our study, galnt11 was used as Single ventricle (morphologic left) 3 (4.8%) a positive control. GALNT11, encoding the polypeptide Single ventricle (morphologic indeterminate) 5 (7.9%) N-acetylgalactosaminyltransferase 11, was previously Other abnormal ventricle arrangement 2 (3.2%) identified as a candidate Htx gene in a patient by CNV Bronchi analysis. The gene plays an important role in the devel- Bilateral right bronchi (short) 34 (54.0%) opment of the LR axis by activating the Notch signaling pathway and modulating the balance between motile and Bilateral left bronchi (long) 7 (11.1%) nonmotile cilia [15, 26]. A standard control provided by Bronchial inversus 22 (34.9%) Gene Tools was injected as a negative control. Spleen Zebrafish embryos were injected with MOs at the Polysplenia 6 (9.5%) one-cell stage, and the direction of heart looping was Asplenia 29 (46.0%) assessed at 48 hpf. We found that MO knockdown of Single right spleen 21 (33.3%) five of the six candidate genes led to altered cardiac looping phenotypes. Compared with the negative control Single left spleen 7 (11.1%) (2.35% abnormality), the phenotypes of five morphants Stomach (numb, pacrg, tctn2, dnah10, and rnf115) differed signifi- Right-sided stomach 38 (60.3%) cantly, with 13.16–26.2% of embryos exhibiting either an Left-sided stomach 19 (30.2%) L-loop or no loop (P < 0.001). However, knockdown of Stomach centrally situated 4 (6.3%) cfap46 had no significant effect on the direction of heart Unknown 2 (3.2%) looping (P > 0.05) (Fig. 3b). Among the five candidate genes, DNAH10 and RNF115 Liver were found to be novel candidate Htx genes in both Left-sided liver 23 (36.5%) humans and animals. dnah10 and rnf115 mutations Liver centrally situated 31 (49.2%) induced by co-injecting zebrafish codon-optimized Cas9 Aortic arch mRNA and dnah10 or rnf115 gRNA also led to a signifi- Left aortic arch 17 (27.0%) cant increase of abnormal heart looping in F0 embryos Right aortic arch 45 (71.4%) (Fig. 3c; P < 0.001), which demonstrates the roles that dnah10 and rnf115 play in LR patterning. Aortic arch centrally descending 1 (1.6%) SVC Over-expression of the candidate genes disturbs cardiac Right SVC 8 (12.7%) looping Left SVC 33 (52.4%) The five candidate genes with phenotypes in MO- Bilateral SVC 22 (34.9%) knockdown embryos were then divided into two groups Liu et al. Genome Medicine (2018) 10:40 Page 6 of 13 Table 2 Nineteen rare copy number variants identified in patients with heterotaxy ID Chromosome Genomic coordinates Type Size (kbp) Genes altered 5 4q24 104,554,264–105,123,728 Internal dup 569.464 TACR3 5 6p22.2 26,019,198–26,227,973 Genic dup 208.775 HIST1H3A, HIST1H4A, HIST1H4B, HIST1H3B, HIST1H2AB, HIST1H2BB, HIST1H3C, HIST1H1C, HFE, HIST1H4C, HIST1H1T, HIST1H2BC, HIST1H2AC, HIST1H1E, HIST1H2BD, HIST1H2BE, HIST1H4D, HIST1H3D, HIST1H2AD, HIST1H2BF, HIST1H4E, HIST1H2BG, HIST1H2AE, HIST1H3E 7 1q21.1 145,625,128–145,927,662 Genic del 302.534 RNF115, CD160, PDZK1, GPR89A, GPR89C, PDZK1P1 10 5q23.1 115,247,380–115,683,172 Genic dup 435.792 AP3S1, AQPEP, LOC644100, COMMD10 16 6p12.1 54,138,106–54,277,341 Genic dup 139.235 TINAG 18 12p13.33 173,786–356,461 Genic dup 182.675 IQSEC3, LOC574538, SLC6A12, SLC6A13 20 12q24.31 123,357,010–124,310,519 Genic dup 953.509 VPS37B, ABCB9, OGFOD2, ARL6IP4, PITPNM2, MIR4304, LOC100507091, MPHOSPH9, C12orf65, CDK2AP1, SBNO1, SETD8, RILPL2, SNRNP35, RILPL1, MIR3908, TMED2, DDX55, EIF2B1, GTF2H3, TCTN2, ATP6V0A2, DNAH10 20 19q13.32 47,308,130–47,418,258 Genic dup 110.128 SNAR-E, AP2S1 26 11q12.2 60,408,411–60,465,698 Genic del 57.287 LINC00301 31 10p15.1 6,254,055–6,374,584 Internal dup 120.529 PFKFB3, LOC399715 34 4q22.2 93,875,432–93,988,049 Genic del 112.617 GRID2 39 8q11.1q11.21 47,398,661–48,407,568 Genic dup 1008.907 LINC00293, LOC100287846, KIAA0146 40 14q24.2 73,620,299–73,786,493 Genic dup 166.194 PSEN1, PAPLN, NUMB 43 4q24 101,476,709–101,668,938 Genic del 192.229 EMCN-IT3 59 2q24.1 157,170,397–157,315,649 Internal dup 145.252 NR4A2, GPD2 59 6q26 163,549,870–163,842,358 Genic dup 292.488 PACRG, PACRG-AS1, DKFZp451B082, CAHM, QKI 59 9p22.2 16,826,417–16,931,236 Internal dup 104.819 BNC2 63 3q25.32 158,198,274–158,256,949 Genic del 58.675 RSRC1 63 10q26.3 134,358,785–134,921,135 Genic dup 562.35 INPP5A, NKX6–2, TTC40, LOC399829, GPR123 Bold items are candidate genes we identified from rare CNVs Genic del deletion of at least one coding exon, Genic dup full duplication of at least one gene, Internal dup duplication of internal exons based on the clinical copy number in the Htx patients: specific heart field effects. Based on the early expression four duplicated genes (numb, pacrg, tctn2, dnah10), patterns of the candidate genes and the patients’ clinical and one deleted gene (rnf115). The functions of the dupli- information of more than one organ malposition, we cated genes were examined using mRNA over-expression hypothesized that the abnormal cardiac looping phenotype analyses. Rescue of the deleted gene, rnf115, was con- results from disruption of early signaling pathways during ducted using an rnf115-pCS2 plasmid. LR development. To test this hypothesis, we examined the Compared with negative control (1% abnormality), expression patterns in zebrafish morphants of pitx2 and over-expression of the candidate genes had a significant lefty2, which are markers of the early common laterality effect on cardiac looping: numb (10.0% abnormality, pathway in the 18–22 somite stage. The pitx2 gene P <0.001); tctn2 (10.2% abnormality, P < 0.001); pacrg encodes a transcription factor that relays LR-patterning (4.95% abnormality, P < 0.05) (Fig. 3d). Over-expression information necessary for proper organogenesis, whereas of the duplicated gene dnah10 could not be carried out lefty2 encodes a protein of the TGF-β superfamily that because its mRNA was too long (14,062 bases). Remark- inhibits nodal activation. The embryonic expression of ably, rnf115 MO knockdown led to 14.3% of abnormal pitx2 and lefty2 exhibited both normal (left side) and cardiac looping, and injection of zebrafish with rnf115 abnormal (right side, bilateral, absent) patterns (Fig. 4a, b). mRNA rescued the normal phenotype (4.7% abnormality). Negative control morphants exhibited approximately 9.1% This shows that this gene has a specific function in LR abnormal pitx2 expression and 16.0% abnormal lefty2 patterning (Fig. 3e). expression. Morphants injected with galnt11 positive control exhibited 31.4% of pitx2 and 37.7% of lefty2 Candidate genes exhibit global effects on early signaling abnormality (P < 0.001). The numb, pacrg, tctn2,and pathways in LR development dnah10 morphants exhibited significant abnormal pitx2 An abnormal cardiac looping pattern can result from and lefty2 expression patterns (20.6–36.2 of pitx2 abnor- either disruption of early common laterality pathways or mality and 27.3–54.7% of lefty2 abnormality; P <0.001). Liu et al. Genome Medicine (2018) 10:40 Page 7 of 13 Fig. 1 Chromosomal view of rare CNVs in candidate Htx patients and the verified results of qPCR. a CytoScan HD array presents a 302.5-kb deletion of 1q21.1 involving RNF115. b A 953.5-kb duplication at 12q24.31 affecting both TCTN2 and DNAH10. In data (a, b), the upper panel depicts log2 ratio data, the middle panel depicts the copy number duplications or deletions, and the lower panel depicts smooth signals of indicated segments. Locations of genes implicated in Htx are shown in the top. Results of qPCR are denoted by yellow stars The rnf115 morphants showed 28.0% abnormality in pitx2 Then, we analyzed the expression of pitx2 and lefty2 expression (P < 0.001) but did not exhibit significant in embryos with over-expression of genes found in genic abnormal lefty2 expression. Consistent with the results duplications (numb, pacrg, tctn2). The results showed above, cfap46 morphants exhibited no significant abnor- that the embryonic expression of pitx2 and lefty2 also malities in either pitx2 or lefty2 expression (Fig. 4c, d). exhibited both normal (left side) and abnormal (right Table 3 Clinical phenotypes of heterotaxy patients with CNVs carrying candidate genes ID Segments Sizes Copy Genes Patients’ cardiac abnormalities Extracardiac abnormalities (kb) numbers 7 1q21.1 (145,625,128–145,927,662) 302.534 1 del RNF115 D, SA, PAVC, LSVC RAA, BI, right spleen, RSS, LSL 20 12q24.31 (123,357,010–124,310,519) 953.509 3 dup TCTN2, DNAH10 D, SA, TA, TGA, PA, VSD, PDA, LSVC RAA, BI, right spleen, RSS, LSL 40 14q24.2 (73,620,299–73,786,493) 166.194 3 dup NUMB D, DORV, PS, VSD, LSVC, SIV RAA, BI, right spleen, RSS, LSL 59 6q26 (163,549,870–163,842,358) 292.488 3 dup PACRG D, SA, SV, MGA, PS, CAVC, LSVC, IRAA LAA, BRB, asplenia, LSS, LCS 63 10q26.3 (134,358,785–134,921,135) 562.35 4 dup TTC40 D, DORV, PS, VSD, PDA, LSVC, SIV RAA, BI, right spleen, RSS, LSL dup duplication, del deletion, PDA patent ductus arteriosus, PS pulmonary stenosis, VSD ventricle septum defect, CAVC complete atrioventricular canal, PAVC partial atrioventricular canal, D dextrocardia, SA single atrium, SV single ventricle, TA tricuspid atresia, PA pulmonary atresia, TGA/MGA translocation of great arteries/ malposition of great arteries, DORV double outlet right ventricle, IRAA isomerism of right atrial appendages, SIV superior-inferior ventricle, LSVC left superior vena cava, RAA right aortic arch, LAA left aortic arch, BI bronchial inversus, BRB bilateral right bronchi (short), RSS right-sided stomach, LSS left-sided stomach, LSL left- sided liver, LCS liver centrally situated Liu et al. Genome Medicine (2018) 10:40 Page 8 of 13 Fig. 2 Whole mount in situ hybridization analysis of candidate genes at two stages: 8–10 somites and primordium 5 stage. a, c, e, g, i, k Results of in situ hybridization of candidate genes and standard control at 13–15 hpf (8–10 somites). Embryos are viewed laterally with anterior to the top to examine KV expression. b, d, f, h, j, l Results of in situ hybridization of candidate genes and standard control at 24 hpf (primordium 5 stage). Lateral view of embryos with anterior to the left.KV (blue arrow), floor plate (red arrows), pronephric duct (green arrows), notochord (yellow arrow), head (black arrows), ubiquitous expression (orange arrows) Liu et al. Genome Medicine (2018) 10:40 Page 9 of 13 Fig. 3 Loss of function of candidate genes in zebrafish disturbed cardiac looping. a Zebrafish heart shows normal dextral loop, abnormal sinistral loop, and no-loop types in cmlc2:eGFP morphants in ventral view. b The percentage of abnormal heart looping with MO injected. The experiments were repeated 3 times, and at each time > 70 embryos were examined for each group. c Summary of the abnormal heart looping of dnah10 and rnf115 mutations generated by co-injection of zebrafish Cas9 mRNA 600 pg and dnah10 gRNA 100 pg or rnf115 gRNA 100 pg. The experiments were repeated 3 times, and at each time > 71 embryos were examined for each group. d Percentage of embryos that exhibit abnormal cardiac looping with mRNA over-expressed. e The rnf115 mRNA can rescue LR randomization. The abnormal heart looping phenotype which is induced by rnf115 MO can be rescued by 6.25 pg rnf115 mRNA. Heart looping direction was assayed in zebrafish at stage 2 dpf. Bars show the total percent of abnormally looped heart including two types: no-loop and sinistral loop heart. Standard control MO (StdCtrl) is negative control. galnt11 is used as positive control. Error bars represent the standard error of the mean (SEM). *P <0.05, **P < 0.01, ***P < 0.001, respectively vs. StdCtrl. WT,wildtype side, bilateral, absent) patterns. Negative control exhibited heterozygous mutations in DNAH10, RNF115, TCTN2, approximately 13.0% abnormal pitx2 expression and 17.7% and NUMB were detected in six sporadic Htx patients abnormal lefty2 expression. Over-expression of galnt11 (Additional file 2: Figure S5 and Additional file 1: positive control exhibited 36.8% of pitx2 and 47.8% of Table S7). lefty2 abnormality (P < 0.001). Over-expression of numb, pacrg,and tctn2 exhibited significant abnormal pitx2 and Discussion lefty2 expression patterns (24.4–40.1% of pitx2 abnormality Htx comprises a class of congenital disorders resulting and 36.9–46.5% of lefty2 abnormality; P < 0.01) (Fig. 4e, f). from malformations in LR body patterning, but the underlying cause in the majority of patients remains Whole-exome sequencing and mutation screening unknown. In an effort to elucidate the molecular mech- In order to further explore the relationship between the anism underlying the pathogenesis of Htx, we recruited candidate genes and Htx, we screened the sequencing 63 children with Htx but free of other syndromes. CNV data of the five candidate genes for rare mutations in analyses identified 19 rare CNVs. Further analyses revealed 65 Htx patients without mutations of other known that six candidate genes associated with the 19 rare CNV laterality-related genes and rare CNVs. Filtering criteria segments were related with pathways reported to be were set as follows: (1) variants are located in exonic or involved in the regulation of LR development. Down- splicing region, (2) exclude synonymous variants, (3) regulation and over-expression of the candidate genes frequency is lower than 0.1% according to public variant in zebrafish demonstrated that five genes (numb, pacrg, databases 1000 Genomes and Exome Aggregation tcnt2, dnah10,and rnf115) strongly affect morphologic Consortium (ExAC), (4) exclude variants detected in our cardiac looping as well as the pattern of pitx2 and lefty2 100 normal Chinese individuals or 2000 non-heterotaxy expression. Moreover, we detected rare mutations in the patients, and (5) at least one scoring software analysis coding sequence of candidate genes DNAH10, RNF115, suggests that mutation is deleterious. Finally, six rare TCTN2,and NUMB in Htx patients. Liu et al. Genome Medicine (2018) 10:40 Page 10 of 13 Fig. 4 Analysis of pitx2 and lefty2 expression patterns in the lateral plate mesoderm in 18–22 somites. a The expression of pitx2 exhibits four patterns, left, right, bilateral, or absent, in the posterior lateral plate mesoderm of zebrafish embryos. b Morphants show left, right, bilateral or absent lefty2 expression in the cardiac field. c, d Summary of abnormal pitx2 and lefty2 mRNA expression in zebrafish morphants. e, f Summary of abnormal pitx2 and lefty2 mRNA expression in zebrafish with mRNA over-expressed. Embryos are viewed dorsally with anterior to the top. Bars show the percent of abnormal pitx2 and lefty2 expression including three types: right, bilateral, absent expression. Standard control (StdCtrl or Std-RNA) is negative control. galnt11 is used as positive control. Error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001, respectively, vs. standard control The study demonstrated that rare CNVs play an import- from 57 to 1009 kb. One duplication segment at 10q26.3 ant role in the pathogenesis of Htx in patients. CytoScan (genomic coordinates 134,358,785–134,921,135, involving HD microarray is a good method for copy-number ana- only five genes) identified in our study was included in lyses. However, balanced chromosomal rearrangements previously published deletion segments associated with such as inversions or balanced translocations could be Htx: a terminal CNV beginning at 10q26.13 [15, 17]. The potentially missed on the CytoScan HD microarray. In remaining 18 segments were never reported. our study, the percentage of subjects with rare CNVs Among the five candidate genes we identified, the (23.7%, 14 of 59 Htx subjects) was higher than that previ- numb, pacrg, and tctn2 genes had been linked to LR ously reported [15, 27]. The size of these rare CNVs varied development in animals but not in humans. The gene Liu et al. Genome Medicine (2018) 10:40 Page 11 of 13 NUMB encodes an endocytic adaptor protein that plays a The only deleted gene identified in our study, RNF115, role in the determination of cell fate. Consistent with pre- also known as breast cancer-associated gene 2 (BCA2), vious reports, zebrafish numb was expressed ubiquitously is a type of E3 ubiquitin ligase. E3 ubiquitin ligases play in the early developmental stage in the present study. important roles in auto-ubiquitination activity, depending Over-expression of numb has been shown to suppress on their RING domain. RNF115 mRNA is expressed at Notch activity, thus causing bilateral distribution of lefty2 moderate levels in the heart, skeletal muscle, and testis . expression and disturbed heart tube looping . This Previous findings indicated that ubiquitination by E3 ligases observation strongly supports our data showing that not regulates a diverse array of cellular functions, such as cilia only over-expression but also downregulation of numb in formation and assembly, and LR development-related zebrafish leads to abnormal cardiac looping and a random signaling pathways (e.g., Nodal signaling) [22–24]. How- distribution of lefty2 and pitx2 expression. ever, the relationship between RNF115 and LR asymmetry The genes pacrg and tctn2 are previously reported to remains unknown. In our study, rnf115 morphant and regulate ciliary function [29–31]. Cilia play a pivotal role mutant zebrafish exhibited disturbed cardiac looping, and in earlier LR development. The gene pacrg is locally rnf115 mRNA rescued the normal phenotype. Moreover, expressed in the KV in zebrafish, where LR asymmetry rnf115 morphants exhibited random distribution of pitx2 is initially established. Xenopus embryos injected with expression but minimal perturbations in the lefty2 expres- pacrg MO exhibit LR laterality defects as well as gastru- sion pattern. According to the previously reported patho- lation and neural tube closure defects [29, 31]. TCTN2 genesis of Htx, asymmetric Nodal signaling activates pitx2 encodes a type I membrane protein of the tectonic family. in the left lateral plate mesoderm via the Smad-FoxH1 TCTN2 was linked to Joubert syndrome, a ciliopathy pathway . Our data suggest that rnf115 may function in disease. Tctn2 knockout mice exhibit ventricular septal the ubiquitination of Nodal-downstream genes (the genes defects and a right-sided stomach, suggesting that Tctn2 in the Smad-FoxH1 pathway) or directly act on pitx2,to plays a role in laterality defects . Our data further regulate LR patterning, which cannot alter the expression show that over-expression of pacrg or tctn2 in zebrafish of lefty2. However, the specific mechanism through also leads to laterality defects. Moreover, this is the first which DNAH10 and RNF115 direct LR axis development report identifying CNVs of NUMB, PACRG,and TCTN2 remains to be further investigated. in patients with Htx. TTC40 encodes cilia and flagella associated protein 46, To date, no reports have linked DNAH10 and RNF115 which is reported to play a role in the occurrence of with LR patterning in either humans or animals. The gene nasopharyngeal carcinoma and acute myeloid leukemia dnah10, expressed in cilia and flagella, is a component of [39, 40]. In our study, zebrafish with knockdown of this the inner dynein arms, which are attached to the periph- gene did not exhibit an abnormal phenotype in terms of eral microtubule doublets; as a protein involved in ATP LR patterning. Moreover, in the patient in our study production, DNAH10 participates in protozoan flagellar with a CNV of CFAP46, Htx might have been caused by motility [32, 33]. Recent studies have demonstrated that a nonsynonymous mutation (c. 841 A > G, p.Trp281Arg) mutations in other cilia dynein heavy-chain genes (such as in LEFTY1 instead. DNAH5, DNAH9,and DNAH11) can cause primary ciliary dyskinesia (PCD). PCD is a severe inherited disorder Conclusions that results from defects in flagellar and ciliary axoneme Our results demonstrate that Htx in some sporadic substructures and is characterized by male infertility, patients maybeattributedtorareCNVs. Moreover,we respiratory diseases, and LR laterality in 50% of affected identified candidate genes in several novel segments of rare individuals [34–36]. We first identified the CNVs of CNVs, some of which have never before been reported as DNAH10 in patients with Htx, and found that knockdown related to LR patterning. Downregulation or over-expression and mutation of dnah10 in zebrafish disturb the LR of the candidate genes in zebrafish disturbed the develop- development and pitx2 and lefty2 expression patterns. ment of LR asymmetry. We believe the results of our study Moreover, we found that dnah10 mRNA is highly advance the understanding of Htx and will aid in its diagno- expressed in the caudal notochord in the primordium 5 sis. However, according to previous literature and the results stage. The notochord breaks bilateral symmetry by of our study [41, 42], we do not have sufficient evidence to altering cell shape and cilia distribution . DNAH10 show that the genes reported in this paper are causal for may regulate LR patterning by affecting the notochord, heterotaxy. They are only candidate genes for Htx. This is which could alter the function of cilia. In addition, both the limitation of our study. The five genes identified in our TCTN2 and DNAH10, located within the same CNV paper are reported for the first time in a heterotaxy popula- segment, can affect the development of LR patterning, tion, and our study can be used as the first evidence for the and whether there is potential interaction between future research of the pathogenic genes of Htx. This is them needs further research. hoped to be further confirmed in other populations. In Liu et al. Genome Medicine (2018) 10:40 Page 12 of 13 addition, other rare CNVs that we did not study might also Ethics approval and consent to participate The research has been performed in accordance with the principles of the play significant roles in development of LR asymmetry. Two Declaration of Helsinki. The study was also approved by the ethics novel candidate genes identified in the present study, committees of Xinhua Hospital (XHEC-C-2012-018, Shanghai, China) and DANH10 and RNF115, should be examined in greater detail Shanghai Children’s Medical Center (SCMC-201004). Parents of the children gave consent for their participation. All zebrafish experiments were with respect to their role in the pathogenesis of defects in conducted at the Institute of Neuroscience, Chinese Academy of Sciences. In LR patterning. the design and process of the zebrafish experiments involved in the study, we have followed relevant guidelines. Additional files Competing interests The authors declare that they have no competing interests. Additional file 1: Table S1. MO sequences, injection doses, and total embryo numbers analyzed for heart looping and gene expression. Publisher’sNote Table S2. Specific primers and vector used to produce genes’ full-length Springer Nature remains neutral with regard to jurisdictional claims in mRNA. Table S3. Antisense RNA probes conducted for whole mount in situ published maps and institutional affiliations. hybridization. Table S4. The frequency of each candidate CNV in normal Chinese individuals and non-heterotaxy patients with developmental delay/ Author details intellectual disability. Table S5. The function of the genes associated with Department of Pediatric Cardiology, Xinhua Hospital, School of Medicine, the 19 rare CNV segments. Table S6. The bioinformatics information on the Shanghai Jiao Tong University, Shanghai, China. The Second Affiliated variant of LEFTY1 in the patient with CNV of TTC40 (CFAP46). Table S7. The Hospital and Yuying Children’s Hospital of Wenzhou Medical University, bioinformatics information on the variants of candidate genes. (PDF 369 kb) Zhejiang, China. Department of Cardiology, Shanghai Children’s Medical Additional file 2: Figure S1. Knockdown efficiency of splice blocking Center, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. and translation blocking MOs in zebrafish embryos. Figure S2. The Scientific Research Center, Xinhua Hospital, School of Medicine, Shanghai knockout efficiency of dnah10 and rnf115 by CRISPR/Cas9. Figure S3. Jiao Tong University, Shanghai, China. Chromosomal view of rare CNVs in candidate Htx patients and the verified results of qPCR. Figure S4. Gene sequencing peak shows a Received: 9 October 2017 Accepted: 10 May 2018 nonsynonymous heterozygous mutation in LEFTY1 in the patient with CNV of TTC40 (CFAP46). Figure S5. Rare variations were detected in Htx patients. (PDF 792 kb) References 1. Jacobs JP, Anderson RH, Weinberg PM, Walters HL 3rd, Tchervenkov CI, Del Duca D, et al. The nomenclature, definition and classification of cardiac Abbreviations structures in the setting of heterotaxy. Cardiol Young. 2007;17 Suppl 2:1–28. CNV: Copy number variant; d-loop: Dextral loop; gRNA: Guide RNA; 2. Taketazu M, Lougheed J, Yoo SJ, Lim JS, Hornberger LK. Spectrum of hpf: Hours post fertilization; Htx: Heterotaxy; kb: Kilobase; KV: Kupffer’s vesicle; cardiovascular disease, accuracy of diagnosis, and outcome in fetal LR: Left-right; Mb: Megabase; MO: Morpholino oligo; PCD: Primary ciliary heterotaxy syndrome. Am J Cardiol. 2006;97(5):720–4. dyskinesia; qPCR: Quantitative real-time polymerase chain reaction; s- 3. Shiraishi I, Ichikawa H. Human heterotaxy syndrome — from molecular loop: Sinistral loop; WMISH: Whole mount in situ hybridization genetics to clinical features, management, and prognosis. Circ J. 2012;76(9): 2066–75. Acknowledgements 4. Zhu L, Belmont JW, Ware SM. Genetics of human heterotaxias. Eur J Hum We are grateful to Dr. J. L. Du for the zebrafish platform support and critical Genet. 2006;14(1):17–25. comments on the experiments. The authors would like to acknowledge Yifan 5. Lin X, Xu X. Distinct functions of Wnt/beta-catenin signaling in KV Zhu for language editing. development and cardiac asymmetry. Development. 2009;136(2):207–17. 6. Bamford RN, Roessler E, Burdine RD, Saplakoglu U, dela Cruz J, Splitt M, et al. Loss-of-function mutations in the EGF-CFC gene CFC1 are associated Funding with human left-right laterality defects. Nat Genet. 2000;26(3):365–9. The project was funded by grants from the National Natural Science 7. Mohapatra B, Casey B, Li H, Ho-Dawson T, Smith L, Fernbach SD, et al. Foundation of China (81270233), the Shanghai Municipal Commission of Health Identification and functional characterization of NODAL rare variants in and Family Planning three-year action plan (GWIV-23), and the Science Committee heterotaxy and isolated cardiovascular malformations. Hum Mol Genet. of Shanghai (13JC1401705). The funding organizations had no role in study 2009;18(5):861–71. design, data collection and analysis, preparation of the manuscript, or in 8. Kosaki R, Gebbia M, Kosaki K, Lewin M, Bowers P, Towbin JA, et al. Left-right the decision to submit the article for publication. axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB. Am J Med Genet. 1999;82(1):70–6. Availability of data and materials 9. Kosaki K, Bassi MT, Kosaki R, Lewin M, Belmont J, Schauer G, et al. The CNV calls presented in Table 2 from Affymetrix CytoScan HD microarray Characterization and mutation analysis of human LEFTY A and LEFTY B, can be accessed through the ArrayExpress repository (https://www.ebi.ac.uk/ homologues of murine genes implicated in left-right axis development. Am arrayexpress/) under accession number E-MTAB-6820. J Hum Genet. 1999;64(3):712–21. The raw data of next generation sequencing of related genes in heterotaxy 10. Kaasinen E, Aittomaki K, Eronen M, Vahteristo P, Karhu A, Mecklin JP, et al. patients generated during the current study can be accessed through the Recessively inherited right atrial isomerism caused by mutations in growth/ Sequence Read Archive (SRA) database [https://www.ncbi.nlm.nih.gov/ differentiation factor 1 (GDF1). Hum Mol Genet. 2010;19(14):2747–53. Traces/study/] under accession number SRP145525. Other data generated or 11. Ware SM, Peng J, Zhu L, Fernbach S, Colicos S, Casey B, et al. Identification analyzed during this study are included in the main paper or its additional and functional analysis of ZIC3 mutations in heterotaxy and related files. congenital heart defects. Am J Hum Genet. 2004;74(1):93–105. 12. Zhian S, Belmont J, Maslen CL. Specific association of missense mutations in Authors’ contributions CRELD1 with cardiac atrioventricular septal defects in heterotaxy syndrome. CL, RC, RX, and KS were involved in conceptualization; CL and RC performed Am J Med Genet A. 2012;158A(8):2047–9. the experiments; FL, SC, and KS provided the clinical information of the 13. Izumi K, Noon S, Wilkens A, Krantz ID. NKX2.5 mutation identification on patients; YX and TL analyzed the sequencing data; CL wrote the main exome sequencing in a patient with heterotaxy. Eur J Med Genet. 2014; manuscript text; KS and RX were involved in supervision, project 57(10):558–61. administration, and funding acquisition. All authors reviewed the manuscript. 14. Sutherland MJ, Ware SM. Disorders of left-right asymmetry: heterotaxy and All authors read and approved the final manuscript. situs inversus. Am J Med Genet C Semin Med Genet. 2009;151C(4):307–17. Liu et al. Genome Medicine (2018) 10:40 Page 13 of 13 15. Fakhro KA, Choi M, Ware SM, Belmont JW, Towbin JA, Lifton RP, Brueckner 38. Burger AM, Gao Y, Amemiya Y, Kahn HJ, Kitching R, Yang Y, et al. A novel M, et al. Rare copy number variations in congenital heart disease patients RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with identify unique genes in left-right patterning. Proc Natl Acad Sci U S A. outcome in invasive breast cancer. Cancer Res. 2005;65(22):10401–12. 2011;108(7):2915–20. 39. Ayadi W, Allaya N, Frikha H, Trigui E, Khabir A, Ghorbel A, et al. Identification 16. Hagen EM, Sicko RJ, Kay DM, Rigler SL, Dimopoulos A, Ahmad S, Mills JL, et al. of a novel methylated gene in nasopharyngeal carcinoma: TTC40. Biomed Copy-number variant analysis of classic heterotaxy highlights the importance Res Int. 2014;2014:691742. of body patterning pathways. Hum Genet. 2016;135(12):1355–64. 40. Saleki R, Christensen T, Liu W, Wang X, Chen QC, Aakre M, et al. A novel TTC40-MSI2 fusion in de novo acute myeloid leukemia with an unbalanced 17. Rigler SL, Kay DM, Sicko RJ, Fan R, Liu A, Caggana M, Mills JL, et al. Novel 10;17 translocation. Leuk Lymphoma. 2015;56(4):1137–9. copy-number variants in a population-based investigation of classic 41. MacArthur DG, Manolio TA, Dimmock DP, Rehm HL, Shendure J, Abecasis heterotaxy. Genet Med. 2015;17(5):348–57. GR, et al. Guidelines for investigating causality of sequence variants in 18. Yu PC, Gu SY, Bu JW, Du JL. TRPC1 is essential for in vivo angiogenesis in human disease. Nature. 2014;508(7497):469–76. zebrafish. Circ Res. 2010;106(7):1221–32. 42. Strande NT, Riggs ER, Buchanan AH, Ceyhan-Birsoy O, DiStefano M, Dwight 19. Xu B, Zhang Y, Du XF, Li J, Zi HX, Bu JW, et al. Neurons secrete miR-132-containing SS, et al. Evaluating the clinical validity of gene-disease associations: an exosomes to regulate brain vascular integrity. Cell Res. 2017;27(7):882–97. evidence-based framework developed by the Clinical Genome Resource. 20. Thisse C, Thisse B, Schilling TF, Postlethwait JH. Structure of the zebrafish Am J Hum Genet. 2017;100(6):895–906. snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development. 1993;119(4):1203–15. 21. Hugh D, Allen DJD, Shaddy RE, Feltes TF. Moss & Adams’ heart disease in infants, children, and adolescents, including the fetus and young adult. Baltimore: Lippincott Williams and Wilkins; 2012. 22. Villumsen BH, Danielsen JR, Povlsen L, Sylvestersen KB, Merdes A, Beli P, et al. A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis. EMBO J. 2013;32(23):3029–40. 23. Cota CD, Bagher P, Pelc P, Smith CO, Bodner CR, Gunn TM. Mice with mutations in Mahogunin ring finger-1 (Mgrn1) exhibit abnormal patterning of the left-right axis. Dev Dyn. 2006;235(12):3438–47. 24. Upadhyay A, Amanullah A, Chhangani D, Mishra R, Prasad A, Mishra A. Mahogunin Ring Finger-1 (MGRN1), a multifaceted ubiquitin ligase: recent unraveling of neurobiological mechanisms. Mol Neurobiol. 2016;53(7):4484–96. 25. Noel ES, Verhoeven M, Lagendijk AK, Tessadori F, Smith K, Choorapoikayil S, et al. A nodal-independent and tissue-intrinsic mechanism controls heart- looping chirality. Nat Commun. 2013;4:2754. 26. Boskovski MT, Yuan S, Pedersen NB, Goth CK, Makova S, Clausen H, et al. The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality. Nature. 2013;504(7480):456–9. 27. Cowan JR, Tariq M, Shaw C, Rao M, Belmont JW, Lalani SR, et al. Copy number variation as a genetic basis for heterotaxy and heterotaxy-spectrum congenital heart defects. Philos Trans R Soc Lond Ser B Biol Sci. 2016;371(1710) 28. Niikura Y, Tabata Y, Tajima A, Inoue I, Arai K, Watanabe S. Zebrafish Numb homologue: phylogenetic evolution and involvement in regulation of left-right asymmetry. Mech Dev. 2006;123(5):407–14. 29. Thumberger T, Hagenlocher C, Tisler M, Beyer T, Tietze N, Schweickert A, et al. Ciliary and non-ciliary expression and function of PACRG during vertebrate development. Cilia. 2012;1(1):13. 30. Sang L, Miller JJ, Corbit KC, Giles RH, Brauer MJ, Otto EA, et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways. Cell. 2011;145(4):513–28. 31. Loucks CM, Bialas NJ, Dekkers MP, Walker DS, Grundy LJ, Li C, et al. PACRG, a protein linked to ciliary motility, mediates cellular signaling. Mol Biol Cell. 2016;27(13):2133–44. 32. Maiti AK, Mattei MG, Jorissen M, Volz A, Zeigler A, Bouvagnet P. Identification, tissue specific expression, and chromosomal localisation of several human dynein heavy chain genes. Eur J Hum Genet. 2000;8(12):923–32. 33. Zukas R, Chang AJ, Rice M, Springer AL. Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei. Biocell. 2012;36(3):133–41. 34. Pazour GJ, Agrin N, Walker BL, Witman GB. Identification of predicted human outer dynein arm genes: candidates for primary ciliary dyskinesia genes. J Med Genet. 2006;43(1):62–73. 35. Schwabe GC, Hoffmann K, Loges NT, Birker D, Rossier C, de Santi MM, et al. Primary ciliary dyskinesia associated with normal axoneme ultrastructure is caused by DNAH11 mutations. Hum Mutat. 2008;29(2):289–98. 36. Bartoloni L, Blouin JL, Pan Y, Gehrig C, Maiti AK, Scamuffa N, et al. Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form of situs inversus totalis and most likely primary ciliary dyskinesia. Proc Natl Acad Sci U S A. 2002;99(16):10282–6. 37. Compagnon J, Barone V, Rajshekar S, Kottmeier R, Pranjic-Ferscha K, Behrndt M, et al. The notochord breaks bilateral symmetry by controlling cell shapes in the zebrafish laterality organ. Dev Cell. 2014;31(6):774–83.
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Published: May 30, 2018