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Introduction

Congenital heart disease (CHD) is the most prevalent birth defect, markedly impacting neonatal health and affecting 2–8% of newborns (1,2). CHD has been attributed to environmental and genetic factors, with the former being considered predominant (3). However, advancements in technology have revealed a higher incidence of CHD in identical twins and individuals with a family history of the condition, and there is growing evidence that genetic factors serve a crucial role in the pathogenesis of CHD (4,5).

It is estimated that 400 genes are associated with CHD, including those encoding transcription factors, cell signaling molecules, and structural proteins involved in heart development, as well as proteins associated with ciliary movement; examples include members of the GATA-binding protein, T-box transcription factor, forkhead box and dynein axonemal heavy chain (DNAH) families (4). The normal function of cilia is necessary for heart development, with key genes involved including those encoding components of the outer and inner dynein arms. Defects in these genes are typically associated with left-right pattern abnormalities and cardiac asymmetry (6). Notable examples include EF hand calcium binding domain (7), kinesin family member 3B (8) and DNAH9. The protein encoded by DNAH9 is an outer dynein arm component crucial for ciliary movement in embryonic nodes. Disruption of DNAH9 can impair human development, potentially leading to abnormal organ alignment along the vertical axis of the body. To date, biallelic mutations in DNAH9 have been identified in 24 patients, who presented with non-syndromic asthenozoospermia (9,10), CHD (10–13), non-syndromic respiratory diseases (10,14–16) and heterotaxy (12,17). Among the CHD patients, only a 14-year-old boy presented with isolated CHD (F6-II in ref.11), and it was unclear whether that patient also had asthenozoospermia (11). Therefore, the relationship between DNAH9 and non-syndromic CHD remains unclear.

The present study identified a compound heterozygous mutation in the DNAH9 gene [NM_001372.3: c.3743+1G>T; c.11176C>T (p.Arg3726Trp)] in a fetus with complex CHD. The c.3743+1G>T mutation is novel and its potential pathogenicity was assessed in vitro. This research expanded the mutation spectrum of the DNAH9 gene and provides a valuable foundation for genetic counseling in families affected by this condition.

Materials and methods

Patients

A naturally conceiving couple comprising a 23-year-old male and a 23-year-old female was recruited for the present study in September 2023 at the Department of Maternity of The First Hospital of Changsha (Changsha, China). Early pregnancy tests, including Down syndrome screening, nuchal translucency screening and noninvasive prenatal testing returned normal results, as did tests for the infectious diseases (HIV, hepatitis B and Treponema pallidum). At 24 weeks and 1 day of pregnancy, an ultrasound examination revealed abnormal sonographic images of the fetal heart, including dextroversion of the heart, complete atrioventricular septal defect, subcardiac total pulmonary venous drainage, pulmonary atresia, arterial catheter blood supply and C-type collateral circulation (Fig. 1), raising the suspicion of complex CHD. The positions of the stomach, liver and spleen of the fetus, a male, were all normal. The pregnant woman later underwent an ultrasound examination at the Obstetrics Department of The Second Xiangya Hospital of Central South University (Changsha, China), and the results were consistent with those obtained at The First Hospital of Changsha. Medical professionals in the Department of Cardiac Surgery of The Second Xiangya Hospital of Central South University (Changsha, China) assessed the situation and recommended early surgical intervention after birth, despite the high surgical risks and uncertain outcomes associated with the procedure. After careful consideration, the pregnant woman and her family chose to terminate the pregnancy and sought to identify a genetic cause. Following an induced abortion, tissue samples from the fetus were collected at The First Hospital of Changsha for copy number variation sequencing and whole exome sequencing (WES) at BGI Genomics Co., Ltd. No chromosomal aneuploidy variants or microdeletions/microduplications known to be clearly pathogenic or suspected to be pathogenic were detected. The present study was approved by the Ethics Committee of The First Hospital of Changsha and the family members signed an informed consent form.

Figure 1. Cardiac ultrasonography of the proband reveals a complex congenital heart disorder. (A) In the four-chamber view of the heart, the apices are oriented toward the right side of the fetus, and the lower portion of the atrial septum and the upper section of the ventricular septum are absent, the width of the defect is ~7 mm. (B) Color Doppler flow imaging confirms the defect. (C and D) Two PVs fail to drain into the atrium. (E) Four PVs form a common vein. (F) The common vein descends and merges with the RPV. (G) The LPV gives rise to a DV and merges with the LHV, MHV and RHV, ultimately draining into the IVC. (H) Blood flow spectrum analysis reveals a flow typically associated with a venous catheter. (I-J) A thick artery is visible on the left side of the heart. It ascends for a considerable distance before (K) branching into a vessel in the neck, which then curves downward and further divides into the RPA and LPA that supply the right and left lungs, respectively. (L) Blood flow spectrum analysis confirms that the vessel is a pulmonary artery. PV, pulmonary vein; AVSD, atrioventricular septal defect; RPV, right branch of the portal vein; LPV, left branch of the portal vein; DV, duct vein; LHV, left hepatic vein; MHV, middle hepatic vein; RHV, right hepatic vein; IVC, inferior vena cava; DA, ductus arteriosus; RPA, right pulmonary artery; LPA, left pulmonary artery.

WES

The genomic DNA of the male proband was extracted from fetal tissue obtained following pregnancy termination using a MagPure Buffy Coat DNA Midi KF Kit according to the instructions provided by the manufacturer (Magen Biotechnology Co., Ltd.). Shearing enzyme (Enzymatics) and magnetic beads (VAHTS DNA Clean Beads; cat. no. N411; Vazyme) were used to fragment and purify genomic DNA to obtain fragments of 200–300 bp. Pre-PCR amplification was then performed to complete library construction. The loading concentration of the final library was 29.2 ng/ml. The DNA of target gene exons and adjacent splicing regions was captured and enriched using the Roche KAPA HyperExome probe set (cat. no. 9718630001; Roche Diagnostics, Ltd.). Gel electrophoresis was used to assess the integrity of DNA, with high-quality DNA displaying a single, clear band. Sequencing Reaction General Kit (T7 SM FCL PE100) v2.0 (cat. no. 1000028455; MGI Tech Co., Ltd.) was used on the MGISEQ-2000 platform (MGI Tech Co., Ltd.) for sequencing. The quality of the raw sequencing data was assessed using SOAPnuke software v2.1.2 (18), and the clean reads were aligned to the hg19 reference genome using Burrows-Wheeler Aligner software BWA-0.7.17 (r1188) (19). Single nucleotide variants and insertions/deletions were identified using the Genome Analysis Toolkit (20), generating results for base polymorphisms in the target region. Subsequently, 1,000 GEA, 1,000 Genomes (East Asian; www.internationalgenome.org/home), gnomAD_exome (East Asian; http://gnomad.broadinstitute.org/) and the Exome Aggregation Consortium (ExAC; East Asian; http://exac.broadinstitute.org), were used to identify the frequency and context of the identified mutations. The BGI-varanno algorithm (21,22) (BGICG_ANNO 0.39; BGI Genomics Co., Ltd) was employed for variant screening and annotation, with the integration of disease data from Clin-Var (https://www.ncbi.nlm.nih.gov/clinvar/), Online Mendelian Inheritance in Man (https://www.omim.org/) and the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/). Variant filtering was performed as described in our previous study (20), and the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology guidelines for mutation pathogenicity were referred to for interpretation (23,24). PCR was performed to amplify the target regions containing the putative variants in the fetus and the parents: DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen GmbH), PCR amplification using GoTaq Green Master Mix (Promega Corporation) under the following conditions: 95°C for 30 sec (denaturation), 59°C for 30 sec (annealing) and 72°C for 30 sec (extension), for a total of 35 cycles, the PCR products were analyzed via gel (2% agarose) electrophoresis to confirm the successful amplification of the target regions. Finally, the PCR products were directly sequenced using Sanger sequencing. The primers used are listed in Table SI.

Bioinformatics analysis

Evolutionary conservation analysis was conducted by aligning the amino acid sequences of DNAH9 proteins across various species using the BLAST tool available on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The potential pathogenicity of the DNAH9 mutations was assessed through in silico analysis utilizing four online tools: Polyphen-2 (http://genetics.bwh.harvard.edu/pph2), MutationTaster (v2021 for GRCh37; http://www.mutationtaster.org/), Sorting Intolerant from Tolerant (SIFT; http://sift.bii.a-star.edu.sg/) and Combined Annotation Dependent Depletion (CADD; v1.7; http://cadd.gs.washington.edu/). The RNA splicing prediction model from the Rare Disease Data Center (RDDC) tool of the Guangzhou Rare Disease Gene Therapy Alliance (https://rddc.tsinghua-gd.org/tool) was used to forecast splicing variations. The mutated sequence was derived from the prediction results. Protein structural analysis of the DNAH9 (NM_001372.3) mutant was performed using the online SWISS-MODEL tool (https://swissmodel.expasy.org), using sequences identified through bioinformatics and minigene experimental products of the splice mutation. Protein structure visualization was performed using PyMol (v3.1.0a0 Open-Source; http://github.com/cgohlke/pymol-open-source-wheels/releases).

Minigene analysis

Due to the absence or low levels of DNAH9 in peripheral blood, obtaining DNAH9 cDNA from this source for splicing pattern analysis was not feasible. Therefore, minigene technology was used to analyze the splicing pattern of the c.3743+1G>T mutation, which is located in intron 19. Bioinformatics analysis using the RDDC tool predicted that this mutation could potentially affect the splicing of exon 19. To investigate the splicing pattern, the target fragment containing exons 18, 19, and 20, along with a partial sequence (150–200 bp) of intron 20, was integrated into a pCMV-MYC vector (Takara Biotechnology Co., Ltd.) using homologous recombination technology. Briefly, primers with homologous arms were designed to amplify the target fragment, which then carried overlapping sequences homologous to the ends of the linearized vector. The integration was achieved through a homologous recombination reaction (ClonExpress MultiS One Step Cloning Kit; Vazyme). Genomic DNA from a control individual and the proband were used as templates to amplify the target sequence, with the necessary primers listed in Table SI. PrimerSTAR MAX DNA Polymerase (Takara Biotechnology Co., Ltd.) was used for amplification under the following thermal cycling conditions: 98°C for 10 sec (denaturation), 58°C for 5 sec (annealing) and 72°C for 30 sec (extension), for a total of 30 cycles. After the plasmid construction was completed, Sanger sequencing was performed on the plasmid. The cells were cultured in a 6-well cell culture plate (CellPro) containing medium composed of 90% DMEM, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin mixture. They were then incubated in a cell culture incubator at 37°C with 5% CO2. When the cell density reached 60–70%, 2 µg of the plasmid DNA was transfected into 293T cells using Neofect DNA transfection reagent (Neofect). At two days following transfection, cellular RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA under the following thermal cycling conditions: 37°C for 15 min, 85°C for 5 sec (PrimeScript RT reagent Kit with gDNA Eraser, Takara Biotechnology Co., Ltd.). The resulting cDNA was then subjected to PCR amplification using GoTaq Green Master Mix (Promega Corporation) under the following conditions: 95°C for 30 sec (denaturation), 59°C for 30 sec (annealing) and 72°C for 30 sec (extension), for a total of 35 cycles. The PCR products were subsequently analyzed by Sanger sequencing. The primers used for amplification are listed in Table SI.

Results

WES and bioinformatics analysis

A compound heterozygous mutation in DNAH9 [NM_001372.3: c.11176C>T (p.Arg3726Trp); c.3743+1G>T] was identified in the fetus by WES and Sanger sequencing. No pathogenic mutations were identified in other genes known to be associated with primary ciliary dyskinesia (PCD). The mother of the proband was a carrier of the c.3743+ 1G>T mutation and the father was a carrier of the c.11176C>T mutation (Fig. 2). The 1000 Genomes Project (East Asian), ESP6500, gnomAD exome and ExAC databases indicate that the c.11176C>T (p.Arg3726Trp) and c.3743+1G>T mutations are extremely rare or absent, respectively, in the general population. Bioinformatics analysis performed using PolyPhen-2, MutationTaster, SIFT and CADD tools predicted both mutations to be pathogenic (Table SII). The c.11176C>T mutation occurs in a residue that is highly conserved across species (Fig. 2C). In addition, this mutation has been previously reported as pathogenic, causing the downregulation of DNAH9 mRNA expression, ultrastructural defects in ciliary outer dynein arms and reduced ciliary beating frequency (11). The c.3743+1G>T mutation is predicted to result in two aberrant splicing events: Skipping of exon 19 (167 bp) and the insertion of a 691-bp segment of intron 19. Compared with the structure of the wild-type DNAH9 protein (Fig. 3A), each aberrant splicing event is predicted to lead to a frameshift and the production of truncated DNAH9 proteins (Fig. 3B). In addition, 3-dimensional (3D) structure prediction indicates that the c.11176C>T (p.Arg3726Trp) mutation will disrupt the hydrogen bond between the arginine residue at position 3,726 and the glutamic acid residue at position 3,594 (Fig. 3C, red arrow). This disruption is expected to lead to the formation of an α-helical structural segment (Fig. 3C, dashed red oval) not present in the wild-type protein (Fig. 3C, dashed blue oval).

Figure 2. Identification of the compound heterozygous mutation in DNAH9 within the family affected by a congenital heart disorder. (A) Pedigree of the family carrying DNAH9 mutations. (B) Locations of the DNAH9 missense mutations identified in the patient and (C) conservation analysis in multiple species. (D and E) Sanger sequencing chromatograms illustrating the DNAH9 mutations in the family affected by the congenital heart disorder. The red arrows indicate the position of the DNAH9 mutations. DNAH9, dynein axonemal heavy chain 9; P1, proband.

Figure 3. Structural models of wild-type and mutant DNAH9, and in vitro minigene analysis of the c.3743+1G>T mutant. (A) Model of wild-type DNAH9 consists of a tail comprising the N-terminus (marked in blue) and a spherical head comprising the C-terminus. The head contains a motor domain (marked in red, green and yellow) and microtubule binding sites (marked in orange). (B) Two predicted protein structures resulting from the splicing of c.3743+1G>T, one of which lacks the N-terminal tail while the other does not have a spherical head. (C) Local structure comparison at p.3726 between the wild-type and c.11176 C>T (p.Arg3726Trp) mutant. In the mutant, the Arg at position 3726 is replaced with Trp, resulting in the loss of a hydrogen bond between Arg-3726 and Glu-3594, and the formation of a new α-helical fragment (shown in the dashed red circle). (D) Results of the in vitro minigene expression experiment show that the c.3743+1G>T mutation produces two abnormal splicing products. One splicing pattern of c.3743+1G>T was the insertion of a 691-bp fragment of intron 19, leading to frameshift and premature termination. The other was the deletion of the 167-bp exon 19, leading to exon jumping, frameshift and premature termination. DNAH9, dynein axonemal heavy chain 9; Arg, arginine; Trp, tryptophan; Glu, glutamic acid; Leu, leucine; Tyr, tyrosine.

Minigene analysis and structure prediction

Minigene analysis revealed that the c.3743+1G>T mutation produced two expression products while the wild-type gene produced only one (Fig. 3D), which is consistent with the results of the bioinformatics analysis. Sanger sequencing confirmed that the product with the higher molecular weight contained a portion of intron 19 (Fig. 3D), while the product with the lower molecular weight resulted from the skipping of exon 19 (Fig. 3D). The 3D structure predictions were consistent with the bioinformatics forecasts, indicating that the two abnormal splicing events lead to the production of two truncated DNAH9 proteins, one lacking a tail and the other missing the spherical head domain (Fig. 3B). According to ACMG guidelines, both the c.3743+1G>T and c.11176C>T (p.Arg3726Trp) mutations are classified as likely pathogenic.

Literature analysis

The 9 previous publications on DNAH9 mutations (9–17), describe findings for 24 patients in total (Table SIII; Fig. S1). These include 10 patients who presented with congenital heart disease (P3, P7-11, P13, P14, P18 and P20), 7 patients with respiratory diseases (P3-6, P21-P23), 3 patients with asthenozoospermia (P1, P2 and P6), and 5 patients who exhibited only situs anomalies (P12, P15-17 and P19). In addition, 1 patient exhibited heterotaxy of abdominal organs and intrauterine fetal death (P24); this patient also carried the RSPH1, c.121G>A (p.G41R) mutation. P6 presented with both respiratory diseases and asthenozoospermia, and P3 had both congenital heart disease and respiratory diseases. Mutation sites associated with different phenotypes were widely distributed across the DNAH9 gene locus, with no obvious pattern discernible.

Discussion

The present study identified a compound heterozygous mutation in the DNAH9 gene associated with structural heart abnormalities. Multiple public databases, including gnomAD, ExAC and the 1000 Genomes Project, indicate that the c.11176C>T (p.Arg3726Trp) and c.3743+1G>T mutations are extremely rare in the general population. The c.11176C>T mutation has previously been reported as pathogenic, and minigene analysis suggests that the c.3743+1G>T mutation is also pathogenic. No other family members of the proband are known to have similar cardiac or other ciliary-related diseases. A multidisciplinary consultation involving cardiac surgeons, geneticists and obstetricians took place to comprehensively evaluate the echocardiographic results of the fetus. The experts unanimously concluded that the cardiac abnormalities of the fetus were highly associated with the DNAH9 gene mutations and recommended genetic counseling and reproductive intervention.

In vertebrates, the fluid flow generated by the ciliary movement of the embryonic node is crucial for the proper positional distribution of organs. Ciliary defects, whether in motile or primary cilia, can impair fluid flow or disrupt left-right positional signaling, leading to abnormal organ distribution (25–30). DNAH9, which is associated with ciliary motility, may cause organ positional abnormalities when defective. The analysis of 24 known cases with DNAH9 mutations in the present study revealed that 3 patients carrying biallelic loss-of-function (LOF) mutations (P16-18) exhibited mild symptoms, such as mirror-image distribution of organs, while 8 patients (P3, P7-11, P13 and P14) carried at least one missense mutation manifesting as CHD. In the present study, a fetus carrying biallelic DNAH9 mutations, specifically a LOF mutation and a missense mutation, also presented with CHD. Therefore, we hypothesize that biallelic LOF mutations, which result in a complete loss of ciliary function, lead to mirror-image organ distribution, whereas other mutations that partially impair ciliary function lead to more severe phenotypes.

Previous research has demonstrated that biallelic mutations in DNAH9 can result in asthenozoospermia, respiratory diseases and CHD. The present study performed an analysis of the mutation sites and phenotypes of DNAH9 mutations for 24 patients reported in nine articles; however, no obvious association between the mutation sites and phenotypes was identified (Fig. S1). This suggests that the function of the DNAH9 gene is complex, and the impact of its mutations on phenotypes may be influenced by multiple factors. However, the small sample size of 24 patients is a limitation of this analysis. Future research integrating gene function studies with analyses of individual genetic backgounds is necessary to more accurately reveal the relationship between mutation sites and phenotypes.

When analyzing the WES results, all genes previously reported to be responsible for PCD were considered as candidate genes, including DNAH9. However, after mutation filtering, only two mutations in DNAH9 were retained. Additionally, ultrasound showed no positional abnormalities of PCD-related organs such as the liver and spleen in the fetus in the present study. However, the possibility that the fetus suffered from syndromic CHD cannot be excluded. Since the fetal respiratory and reproductive systems were not yet fully developed, it was not possible to assess the impact of the mutations on these two systems through existing clinical means. Therefore, it is necessary to develop novel strategies to verify the effects of mutations on these two systems. The lack of a differential diagnosis to rule out other potential ciliary dysfunctions is a limitation of the present study.

Congenital heart defects resulting from genetic abnormalities can be severe and often require medical management and surgical intervention; they can even be life-threatening, imposing a significant financial and psychological burden on families. Reproductive interventions, such as preimplantation genetic testing (PGT) and prenatal diagnosis, are essential tools for preventing and managing birth defects by helping to avoid the birth of children with CHD caused by genetic factors. The DNAH9 protein is an outer dynein arm protein that serves a crucial role in the movement of embryonic nodal cilia and is essential for cardiac development. The present study identified DNAH9 mutations as the underlying cause of CHD in a specific family, providing the couple with the opportunity to have a healthy child through PGT or prenatal diagnosis.

Supplementary Material

Supporting Data

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by grants from the open research fund of National Key Research and Development Program of China (grant no. 2023YFC2705605), Hunan Provincial Key Laboratory of Regional Hereditary Birth Defects Prevention and Control (grant no. HPKL2023032), the National Natural Science Foundation of China (grant no. 82201773), Hunan Provincial Natural Science Foundation (2023JJ30716) and the Hunan Provincial Science and Technology Innovation Plan Project (grant no. 2021SK53204).

Availability of data and materials

The original data generated using whole-exome sequencing in this study have been deposited into Mendeley Data (V1) with the accession number doi: 10.17632/tgdng3bt9y.1 (https://data.mendeley.com/datasets/tgdng3bt9y/1). In addition, the data generated in the present study may be requested from the corresponding author.

Authors' contributions

JY and CYY were responsible for performing clinical work, recruiting the patients and obtaining informed consent. HYZ conducted the ultrasound examinations. JY and WBH conceived and designed the experiments, while XL and JLZ performed the experiments. XL authored the primary manuscript, while JY and WBH reviewed and revised the manuscript. WBH also acquired funding. All authors read and approved the final version of the manuscript. JY and WBH confirm the authenticity of all the raw data.

Ethics approval and consent to participate

This study was approved by the institutional ethics committee of The First Hospital of Changsha (Changsha, China), and written informed consent was obtained from all participants.

Patient consent for publication

Patient consent for publication was obtained from all participants.

Competing interests

The authors declare that they have no competing interests.

References