Solitary patent ductus arteriosus in a Japanese macaque (Macaca fuscata): a case report and genetic investigation

doi:10.21203/rs.3.rs-2757104/v1

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Solitary patent ductus arteriosus in a Japanese macaque (Macaca fuscata): a case report and genetic investigation

https://doi.org/10.21203/rs.3.rs-2757104/v1

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We present the first report of solitary patent ductus arteriosus (PDA) without associated cardiovascular anomalies in a Japanese macaque (Macaca fuscata). The macaque showed no signs of PDA during its lifetime and lived for 9 years until the PDA was discovered post-mortem. An hourglass-shaped PDA with a total length of 19.8 mm and minimal diameter of 6.7 mm was observed using a silicon casting. Whole-exome sequencing was employed to investigate possible genetic factors related to PDA, and five missense candidate single-nucleotide variants (SNVs) were detected in ADAM15, AZGP1, CSPG4, EPOR, and TNFRSF13B. The conserved nature of the SNV sites in these genes across reference primate species, excluding the macaque, suggests that these mutations may play a role in PDA in macaques. This case report provides new information about solitary PDA in Japanese macaques and broadens the scope of non-human primate research through genetic investigation.

Japanese macaque

patent ductus arteriosus

single-nucleotide variant

whole-exome sequencing

Patent ductus arteriosus (PDA) is a rare condition in monkeys documented in only a few species (Freigang and Knobil 1967; Jerome 1987; Peterson et al. 1997). In contrast, PDA is a common congenital heart defect in humans, accounting for 5–10% of all congenital heart disease cases (Dice and Bhatia 2007). Despite the difference in the incidence of PDA and the similarities between the cardiovascular systems of humans and monkeys (Cox et al. 2017), no research focused on PDA in monkeys has been conducted.

The contribution of genetic factors to PDA remains incompletely understood (Lewis et al. 2018). While PDA was once thought to be a sporadic disease, recent studies have demonstrated a high familial incidence, prompting investigations into potential genetic factors associated with the condition (Bhandari et al. 2009). Studies of patients with syndromic PDA, including Char (TFAP2B), Mowat-Wilson (ZEB2), Noonan (PTPN11), and Holt-Oram (TBX5) syndromes (Hajj and Dagle 2012), have shed light on the genetic mechanisms underlying PDA. The development of whole-exome sequencing (WES) has allowed researchers to actively explore the genetic causes of non-syndromic PDA, with recent studies uncovering rare damaging variants in six candidate genes (SOX8, NES, CDH2, ANK3, EIF4G1, and HIPK1) as potential causes of PDA (Gao et al. 2022).

We present the first report of solitary PDA in a Japanese macaque (Macaca fuscata) without any other congenital cardiovascular anomalies. We described the shape of the PDA in this animal using a vascular cast of the aorta. To investigate potential genetic variants related to PDA, we conducted whole exome sequencing (WES) on the DNA of the Japanese macaque.

This case study investigated a 9-year-old male Japanese macaque that resided in a zoo in Busan, Korea, for exhibition purposes from 2015 to 2021. No evidence of PDA was detected during the lifetime of the monkey. The macaque became weak and fatigued despite regular food intake. Peripheral edema initially developed in the extremities before extending to the abdomen and rest of the body, which constrained mobility. The animal experienced significant weight loss, making the dorsal spinous processes along the spine easily palpable. Although necropsy identified chronic liver failure and cirrhosis as the primary etiology, the PDA remained undetected.

The cadaver was transported to the Department of Veterinary Anatomy at Jeonbuk National University to prepare a silicone cast and examine the aortic arch branching pattern, as described previously (Ahn et al. 2008). Briefly, silicone (Lucky-Silicon; Wacker Chemical Korea Co. Ltd., Jincheon, Republic of Korea) was injected into the cranial side of the celiac artery. During this process, we unexpectedly discovered a PDA connecting the descending aorta and main pulmonary artery, as the silicone entered the main pulmonary artery via the PDA in an abnormal manner (Fig. 1). During dissection of the heart, we did not detect any other congenital heart defects, such as a ventricular or atrial septal defect.

After examining the silicone cast, we identified an hourglass-shaped PDA linking the descending aorta and main pulmonary artery. The PDA featured a central constriction and two ampullae—one on the aortic side and the other on the pulmonary side (Fig. 1). We measured the length and diameter of blood vessels within the silicone cast to investigate the morphological characteristics of the PDA. The minimum diameter of the PDA was 6.7 mm, with the pulmonary end measuring 16.4 mm and the aortic end 15.0 mm. The length of the pulmonary ampulla (from the pulmonary end to the constriction of the PDA) was 8.1 mm, while the length of the aortic ampulla (from the aortic end to the constriction) was 11.8 mm. The full length of the PDA, from the pulmonary to the aortic end, was 19.8 mm. We measured the diameters of the aorta and main pulmonary artery perpendicular to their longitudinal axes. The diameters of the proximal and distal levels connecting the aortic end of the PDA were 15.0 and 11.1 mm, respectively. The diameter of the main pulmonary artery at the bifurcation level was 17.8 mm. The right and left pulmonary arteries measured 12.4 and 12.5 mm in diameter below the bifurcation, respectively.

Using the Illumina sequencing platform (Illumina, San Diego, CA, USA), WES was performed on the macaque’s DNA by Macrogen (Seoul, South Korea) to investigate genetic candidates related to PDA. The exome was isolated using a human exon capture kit, i.e.,- the SureSelect V6+UTR-post kit (Agilent Technologies, Inc., Palo Alto, CA, USA). The resulting raw sequencing data were aligned to the Mmul_10 Macaca mullata reference genome assembly from the National Center for Biotechnology Information (NCBI) using the BWA-MEM mapping program (version 0.7.1). Variants, including single-nucleotide variants (SNVs) and indels, were identified and annotated using SnpEff software (version 4.3). Of the 6,227 identified variants, we applied a filter based on the annotation of variants, resulting in 1,133 variants for further analysis. We filtered the candidate SNVs using publicly available databases, including OMIM, MalaCards, ClinVar, STRING, the Gene Ontology database, and relevant literature, to assess their potential relevance to PDA and cardiovascular development (Gao et al. 2022). This process resulted in a final list of 32 potentially relevant variants.

We employed the nucleotide BLAST program available on the NCBI platform to compare the 32 potentially relevant SNVs with sequences from five reference species, including Macaca fuscata, Macaca mulatta, Macaca fascicularis, Papio anubis,and Homo sapiens. This comparison was performed to assess the conservation level across species and identified the potential functional consequences of these variants. We focused on SNVs unique to the subject macaque (i.e., absent from all five reference species). Consequently, we observed five missense candidate SNVs, in the ADAM15, AZGP1, CSPG4, EPOR, and TNFRSF13B genes. We detected a heterozygous SNV in the ADAM15 gene at position Chr1:95,974,333C>G, resulting in a XM_015110966.2:c.1653G>C, p.Gln551His change in exon 14. The AZGP1 gene presented a homozygous SNV at position Chr3:42,137,536T>C, leading to a NM_001194114.1:c.770A>G, p.Asn257Ser alteration in exon 4. We observed a heterozygous SNV at position Chr7:52,823,053T>C in the CSPG4 gene, which caused a XM_028851149.1:c.2528A>G, p.Asp843Gly change in exon 3. The EPOR gene exhibited a heterozygous SNV at position Chr19:11,141,681C>G, resulting in a XM_001105833.4:c.307G>C, p.Val103Leu change in exon 3. Lastly, we identified a heterozygous SNV at position Chr16:16,763,921G>A which led to a XM_015118722.2:c.878C>T, p.Ala293Val alteration in exon 5 (Fig. 2).

To elucidate the potential functional relationships among proteins affected by the five identified SNVs and those associated with PDA or a congenital heart defect, we generated a protein-protein interaction (PPI) network using the Cytoscape program (version 3.7.0) (Fig. 3). The PPI network was constructed based on information from the STRING database, a comprehensive resource for known and predicted protein interactions. In the network, candidate genes including AZGP1, CSPG4, EPOR, and TNFRSF13B showed direct or indirect relationships with known pathogenic genes in both groups, whereas ADAM15 did not.

This case report is significant because it is the first to describe solitary PDA in a Japanese macaque without any other associated cardiovascular anomalies. Research on PDA in monkeys remains scarce and only a few cases have been reported in various species, including stump-tailed and rhesus monkeys, along with cases of both atrial septal defects and PDA in cynomolgus and rhesus monkeys, and of both ventricular septal defects and PDA in cynomolgus and African green monkeys (Freigang and Knobil 1967; Jerome 1987; Peterson et al. 1997). Koie et al. reported a case of tetralogy of Fallot in a Japanese macaque, which is characterized by severe right ventricular hypertrophy, a congenital ventricular septal defect, an overriding aorta, and stenosis of the pulmonary valve (2007). Although this case also involved a PDA, it was not a solitary PDA.

The low incidence of PDA in monkeys underscores the significance of our report. Data on the incidence of PDA in monkeys is limited; a study by Peterson et al. identified only one case of PDA among 4,390 rhesus monkeys, indicating a low incidence of PDA in this species (1997). This contrasts with the relatively high incidence of 1 in 2,000 observed in humans; no clear explanation has been provided for this disparity in incidence. Focusing research on PDA in monkeys may provide valuable insights into why this condition is rare in monkeys but common in humans.

A noteworthy aspect of this report is that the PDA in the Japanese macaque remained undiagnosed until postmortem. The macaque did not exhibit any symptoms related to PDA and survived for 9 years without difficulties. A ductal size > 3.0 mm is deemed indicative of severe PDA (McNamara and Sehgal 2007). However, in this case report, the macaque had a ductal size of 6.7 mm. Despite accounting for the arterial flexibility associated with the pressure of the silicone injection, the ductal size was quite large, although the macaque did not exhibit any PDA-related symptoms. We suspect that the limited mobility and sedentary lifestyle of monkeys in zoos may lead to undiagnosed cases; mild cases of PDA may go undetected as there is no need to engage in activities that could potentially exacerbate the symptoms. Therefore, increased attention may be necessary to identify PDA in monkeys in zoos.

Genetic factors contributing to PDA in humans remain incompletely understood, and research on PDA in animals is even more limited. Considering that PDA is found in most mammals, broadening the research scope to include various animal species would advance our understanding of PDA. Monkeys, in particular, have highly similar physiological and pathological systems to humans, and also have genetic similarities; studying PDA in these species is therefore of significance (Cox et al. 2017).

Technical challenges arise when conducting genetic research in monkeys. Although WES is a powerful tool to study genetic factors associated with congenital disease, its application to monkeys is limited by the current lack of a species-specific exon capture kit for their genomes. Several studies have reported use of human exon capture kits in non-human primates, owing to the genetic similarity between humans and monkeys (Chan et al. 2021); however, the application of this method for investigating disease-related genetic factors in monkeys remains unexplored. Our report represents the first attempt to apply this approach, emphasizing its potential value for investigating other as-yet unknown genetic diseases in monkeys.

We identified five missense candidate SNVs in the macaque, including ADAM15, AZGP1, CSPG4, EPOR, and TNFRSF13B. ADAM15 is involved in cell-cell and cell-matrix interactions associated with cell adhesion, migration, and proteolysis (White 2003). AZGP1, a secreted protein implicated in lipid metabolism and regulation of the cell cycle, exhibits downregulation during ductal closure (Ovalı 2020). CSPG4, a transmembrane proteoglycan expressed in multiple cell types, including pericytes, plays a crucial role in blood vessel development and stability (Stallcup 2018). EPOR is expressed on the surface of erythroid progenitor cells and some non-hematopoietic tissues, including vascular endothelial cells (Trincavelli et al. 2013). TNFRSF13B, a receptor critical for the development and survival of B cells (Rickert et al. 2011), exhibits a NM_012452.3:c.310T > C:p.Cys104Arg variant of uncertain significance (-) in a patient with PDA, as reported in the ClinVar database. While the direct role of these genes in PDA development requires further validation, our findings reveal that their SNV sites are conserved across five reference primate species, with the exception of the macaque, implying that these unique mutations may potentially contribute to PDA.

A limitation of this case report is that it involved only a single case, which presents statistical constraints. However, PDA is extremely rare in macaques, which made it difficult to secure a large sample. Therefore, a worldwide collaborative research effort is necessary to better understand PDA in macaques; it is hoped that reporting this case prompts such an effort and further investigation into this rare condition.

In conclusion, this is the first case report of a solitary PDA in a Japanese macaque without any other associated cardiovascular anomalies. The rarity of PDA in monkeys highlights the importance of this report. In addition, the genetic investigation conducted that we conducted broadens the scope of non-human primate research of PDA.

Acknowledgements

This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (MSIT), Government of the Republic of Korea (grant number: NRF-2019R1I1A3A01064272). The authors would like to express their sincere appreciation to the participating veterinarian who contributed valuable information about the subject.

Funding

Competing interest statement

The authors declare no competing interests.

Author Contributions

The study's conceptualization was contributed by Dongchoon Ahn and Jeongha Sim, who also provided overall supervision. Young-Jin Jang was responsible for preparing the materials, analyzing the data, and interpreting the results. Byung-Yong Park and Hyun-Jin Tae assisted with analyzing the whole exome sequencing data. The initial draft of the manuscript was prepared by Young-Jin Jang, and all authors contributed to revising and commenting on previous versions. The results of the study were discussed by all authors, and they approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.

Ethics approval

The current study exclusively an employed animal carcass. The Institutional Animal Care and Use Committee (IACUC) at Jeonbuk national university has confirmed that no ethical approval is required.

Consent to publish

Not applicable.

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No competing interests reported.

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Solitary patent ductus arteriosus in a Japanese macaque (Macaca fuscata): a case report and genetic investigation

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