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Article

Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies

by
Adel Abuzenadah
1,2,
Saad Alsaedi
3,
Sajjad Karim
1,* and
Mohammed Al-Qahtani
1
1
Center of Excellence in Genomic Medicine Research, Faculty of Applied Medical Sciences, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia
2
King Fahd Medical Research Center, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia
3
Department of Pediatric, Faculty of Medicine, King Abdulaziz University Hospital, King Abdulaziz University, P.O. Box 80215, Jeddah 21589, Saudi Arabia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(11), 3547; https://doi.org/10.3390/ijms19113547
Submission received: 15 September 2018 / Revised: 1 November 2018 / Accepted: 5 November 2018 / Published: 10 November 2018
(This article belongs to the Special Issue Transcriptional Regulation: Molecules, Involved Mechanisms and Misregulation)
Browse Figures
Versions Notes

Abstract

:
Patau Syndrome (PS), characterized as a lethal disease, allows less than 15% survival over the first year of life. Most deaths owe to brain and heart disorders, more so due to septal defects because of altered gene regulations. We ascertained the cytogenetic basis of PS first, followed by molecular analysis and docking studies. Thirty-seven PS cases were referred from the Department of Pediatrics, King Abdulaziz University Hospital to the Center of Excellence in Genomic Medicine Research, Jeddah during 2008 to 2018. Cytogenetic analyses were performed by standard G-band method and trisomy13 were found in all the PS cases. Studies have suggested that genes of chromosome 13 and other chromosomes are associated with PS. We, therefore, did molecular pathway analysis, gene interaction, and ontology studies to identify their associations. Genomic analysis revealed important chr13 genes such as FOXO1, Col4A1, HMGBB1, FLT1, EFNB2, EDNRB, GAS6, TNFSF1, STARD13, TRPC4, TUBA3C, and TUBA3D, and their regulatory partners on other chromosomes associated with cardiovascular disorders, atrial and ventricular septal defects. There is strong indication of involving FOXO1 (Forkhead Box O1) gene—a strong transcription factor present on chr13, interacting with many septal defects link genes. The study was extended using molecular docking to find a potential drug lead for overexpressed FOXO1 inhibition. The phenothiazine and trifluoperazine showed efficiency to inhibit overexpressed FOXO1 protein, and could be potential drugs for PS/trisomy13 after validation.

1. Introduction

Patau Syndrome (PS) is a rare congenital anomaly due to the presence of an extra chromosome 13 popularly called trisomy 13 [1]. In spite of being the least common, it is the severest of all autosomal trisomies indicated by a prevalence rate of 1:5000 to 1:20,000 [2,3]. The syndrome is associated with a host of congenital anomalies including central nervous system (CNS) defects, midline abnormalities, eye and ear anomalies, cardiac defects, apnea, orofacial flaws, gastrointestinal and genitourinary aberrations, limb deformations, and developmental retardation [4,5]. Life expectancy is severely limited; more than 80% of PS patients do not survive long, and according to some estimates have median survival of 2.5 days [2,6,7]. Nevertheless, only a few can survive beyond 10 years but not with serious intellectual and physical disabilities [8,9,10,11]. Early death of PS is assigned to frequent CNS and cardiopulmonary system aberrations [12].
There is no specific treatment recommended for PS. Intensive care unit level of treatment for a couple of weeks is requisite for infants. Surgery for heart defects and other abnormalities like gastrointestinal or urogenital might be needed for six-month survivors. However, CNS disorders are difficult to treat by surgery. Children surviving more than a year suffer from severe intellectual disabilities, physical abnormalities and also have a high risk of developing cancer. Most studies indicate that older women are at higher risks of delivering trisomy 13 offspring [13]. Despite the fact that there are a number of trisomy 13 cases in Saudi Arabia, no systematic study has yet been done on causative factors like maternal age, consanguinity, and parity.
PS is a multigenic complex and lethal disease of multiple congenital abnormalities associated with poor prognosis [14]. Along with CNS disorders, heart ailments, especially septal defects are leading cause of deaths [2,15]. Septal defects is a complex disorder involving hundreds of altered gene regulations and these genes are located on multiple chromosomes including chromosome 13 [16]. Chromosome 13 is 114,364,328 bp in size, representing nearly 4% of the total DNA, and encodes 308 proteins. This chromosome has 343 protein-coding genes, 622 non-coding RNA genes, and 481 pseudogenes [17].
Molecular pathway and gene ontology analysis of chromosome 13 revealed the presence of important genes like FOXO1, Col4A1, HMGBB1, FLT1, EFNB2, EDNRB, GAS6, TNFSF1, STARD13, TRPC4, TUBA3C, TUBA3D. These genes are linked with cardiovascular disorders, atrial and ventricular septal defects commonly reported in PS [18,19,20,21,22,23,24,25,26,27,28,29,30,31]. Among them, FOXO1 is a strong transcription factor which interacts and regulates several other genes on different chromosomes, (GATA4 (8p23.1), GATA6 (18q11.2), GJA1 (6q22.31), JAG1 (20p12.2), CITED2 (6q24.1), RYR2 (1q43), NKX2-5 (5q35.1), RARA (17q21.2), CXCL12 (10q11.21), SIRT1 (10q21.3), TBX5 (12q24.21), AKT1 (14q32.33), CDKN2A (9p21.3), PCK1 (20q13.31), etc.) and are associated with septal defects in PS [32,33,34,35,36,37,38,39,40,41,42,43,44,45]. Thus, some genes like NODAL, FPR1, AFP, AGO2, UROD, ZIC2 are not located on chromosome 13 but have strong association with PS.
Forkhead Box O1 (FOXO1) gene needs special mention. It is a member of the forkhead box O family of transcription factors located on 13q14.11. The FOXO1 exhibits its functions by binding to promoter of downstream genes or interacting with other transcription factors [46]; both its up- or down-regulation can lead to serious consequences. It has noticeable expression in the cardiovascular system, specifically in vascular and endothelial cells, and plays a substantial role in the crucial embryonic stage [22,47]. The specific function of FOXO1 has to be determined. However, some studies strongly suggest its key role in regulation of numerous cellular functions comprising proliferation, survival, cell cycle, metabolism, muscle growth differentiation, and myoblast fusion [48,49,50]. Other observations relate it to muscle fiber-type specification highly expressed in fast twitch fiber-enriched muscles, in comparison to slow muscles. The FOXO1 is also involved in a host of other functions: metabolism regulation, cell proliferation, oxidative stress response, immune homeostasis, pluripotency in embryonic stem cells, and apoptosis [51,52]. Besides, FOXO1 deletion or downregulation helps to rescue heart from diabetic cardiomyopathy and increases apoptosis under stress conditions like ischemia or myocardial infarction [52,53,54,55]. The FOXO1 is a major transcription factor in cardiac development. Thus, we see FOXO1 null mice have underdeveloped blood vessels, whereas overexpression of the FOXO1 gene results in reduced heart size, myocardium thickening, and eventual heart failure [18,19,20,21]. Since FOXO1 protects cardiac tissue from a variety of stress stimuli by up-regulating anti-apoptotic, antioxidant, and autophagy genes [47,56,57], and restores metabolic equilibrium to minimize cardiac injury due to apoptosis, therefore, in PS, FOXO1 might be a chief regulator of cardiac disorders [52]. The fact is reinforced by reports where survival is improved by suppression of upregulated FOXO1 [18]. Given the wide range of functions of FOXO1, its expression rate may play a vital role in PS and we checked its inhibition via molecular docking with certain drugs.

Molecular Docking between Candidate Drugs and FOXO1

Molecular docking, a computational simulation to screen inhibitor (ligand) compounds against biomolecule of interest, has become a crucial aspect of drug discovery approaches. Recently, repositioning or repurposing of the existing drugs is gaining attention for the treatment of diseases other than their known primary indications [58,59]. This approach could save enormous time, efforts and costs owing to the proven safety and quality of the drugs already available on the market, rather than to discover and develop novel chemical leads [60]. Similar observation on FOXO1, already implicated in a variety of functions, can specially be very promising for docking studies.
The FOXO1 protein contains 4 functional domains; (i) Forkhead domain (FKH), (ii) nuclear localization signal domain (NLS), (iii) nuclear export signal (NES), and (iv) transactivation domain (TAD). The FKH domain consists of four helices (H1–H4), two winged-loops (W1–W2), and three β strands (S1–S3), which mainly exhibits its functions as a DNA recognition and binding site. The FOXO1 regulates transcription of genes by directly binding with either 5′-GTAAA(T/C)AA-3′, or 5′-(C/A)(A/C)AAA(C/T)AA-3′ consensus sequence of downstream DNA [61,62,63]. The FOXO1 protein has thus become an extremely useful therapeutic target in many diseases including PS. Its expression can be regulated by acetylation, phosphorylation, and ubiquitination. Many potential inhibitors including leptomycin B [64], phenothiazines/trifluoperazine [65,66], bromotyrosine/psammaplysene A [67] or D4476 [68] and ETP-45658 [69], have been identified via virtual screening. Some drug candidates directly targeting FOXO1 have been patented [66]. For the docking study, we picked the FDA-approved drugs phenothiazine and its derivatives, trifluoperazine, which binds directly to the DNA binding domain of FOXO1 [70,71]. A brief introduction of both will be befitting here.
Phenothiazine (PTZ) and its derivatives are organic antihelmintic compounds presently used for important diseases like schizophrenia and bipolar disorder. Dopamine receptors are their main target. Repurposing PTZ has been tried earlier for developing novel antitumor agents [72] and Hepatitis C virus [73]. Trifluoperazine (TFP), the other derivative chosen in our studies, is a phenothiazine derivative and a dopamine antagonist, with antipsychotic and antiemetic properties. Their scaffold derivatives have also been suggested as an antiglioblastoma agent [74] and chemotherapeutic anticancer agent with high efficacy and reduced toxicity especially for oral cancer [72]. Lately, they have been shown as calmodulin antagonist [75,76].
In view of the fact that the exact mechanism is unknown as to how trisomy 13 disrupts development, heart disorders were identified as one of the most common disorders causing early death of PS patients. The present study, therefore, aims to explore the molecular interactions of 308 genes on this chromosome. We describe here the distinctive function of chromosome 13 and its key genes, especially FOXO1. We further intended to design a potential drug against FOXO1, a strong transcription factor which interacts with other key genes associated with lethal heart disorders in PS. The potential drugs to inhibit/reduce the transcriptional factor properties of FOXO1 are further explored with an aim to restore metabolic balance and limit apoptosis-induced cardiac damage.

2. Results

2.1. Cytogenetic Analysis of PS Patient

The prime aim of the current work was conducting genetic analysis of PS cases in the Saudi society (n = 37). Cytogenetic analyses were performed using G-banding technique-based karyotyping and found “full trisomy 13” in all 37 PS cases (Figure 1). The majority of individuals were newborns or children (up to 2 years), all with multiple abnormalities including heart disorders. Male to female ratio was found as 1.2:1. Analysis showed that mothers of affected individuals were above 35 years. The key clinical findings of PS observed: congenital heart defects (CHD) (61%), dysmorphic features (56%), polydactyly of hands and/or feet (53%), cryptorchidism (51%), abnormal auricles/low-set ears (47%), microphthalmia (40%), neurological disorders/microcephaly (35%), micrognathia (33%), scalp defects (31%), oral clefts (17%), microphthalmia/anophthalmia (9%), and duplication of the hallux (3%). Out of 37 cases, 31 underwent echocardiography and/or ultrasound, 21 of them showed heart defect and asymmetry of cardiac chambers. The main anatomical defects observed were arterial or ventricular septal defect, patent ductus arteriosus, pulmonic stenosis, coarctation of the aorta, tricuspid valve regurgitation, and mixed defects.

2.2. Molecular Pathway Analysis

Diploid status of chromosome 13 and normal expression of its genes are vital and a number of diseases are associated with its abnormalities (Table S1). However, molecular pathway and gene ontology analysis show as many as 308 protein coding genes on chr13; some of these pathogenic genes are ATP7B, BRCA2, CAB39L, CKAP2, ESD, GJB2, GJB6, GPC5, HTR2A, MBNL2, RB1, SOX21, ZMYM2, collectively noted for various disease associations. Other important genes such as Col4A1, EFNB2, EDNRB, FLT1, FOXO1, GAS6, HMGB1, STARD13, TRPC4, TUBA3C, ZIC2 are specifically associated with cardiovascular disorders, atrial and ventricular septal defects—the key disorders of PS (Table 1). Ingenuity pathway analysis on 308 genes revealed canonical pathways like estrogen-mediated S-phase entry (Figure 2), gap junction signaling, cancer signaling, nitric oxide signaling in the cardiovascular system, adipogenesis pathway, VEGF signaling, cell cycle: G1/S checkpoint regulation, angiopoietin signaling, and 14-3-3-mediated signaling (Table 2). For a comprehensive idea, canonical pathways based on protein coding genes are summarized in Table 2. A cursory look shows FOXO1 to be involved in most of the canonical pathways. We focused our attention on it being strong transcription factor, interacting with and regulating many other genes on different chromosomes associated with septal defects in PS.

2.3. Genomic Analysis and Protein–Protein Interaction Study

The result of STRING displayed direct interaction and predicted functional relationship amid FOXO1 and its interacting proteins. The following proteins showed noticeable interactions with FOXO1: GATA4 (8p23.1), SIRT1 (10q21.3), CITED2 (6q24.1), NFATc1 (18q23), and TBX5 (12q24.21) (Figure 3). FOXO1 as transcription factor interacted with the following relevant target genes: FASLG (1q24.3), IGFBP1 (7p12.3), SOD2 (6q25.3), PPARGC1A (4p15.2), ADIPOQ (3q27.3), APOC3 (11q23.3), OSTN (3q28), BCL2L11 (2q13), CCND2 (12p13.32), and CDKN1B (12p13.1). This was predicted by text-mining application and UCSC genome browser. However, genomic analysis of PS had shown that many genes (NODAL on 10q22, FPR1 on 19q13.41, AFP on 4q13.3, AGO2 on 8q24.3, UROD on 1p34.1, ZIC2 on 13q32.3, etc.) are not directly regulated by FOXO1, rather strongly associated with PS (Table 3).
Docking using the Lamarckian Genetic Algorithm approach was employed to elucidate the basis of structural binding of PTZ and TFP to FOXO1. The result demonstrated favored binding energies ΔG in the range of −4.17 kcal/mol to −1.87 kcal/mol, respectively, with 1 molecule of PTZ showing hydrogen bond with the active site residue Ser193. Other predominant interactions for PTZ were hydrophobic (Leu163, Leu168, Val194, and Pro195) and pi–pi ring stacking non-covalent interaction with Trp189. Estimated inhibition constant, Ki values were 879.98 µM (FOXO1:PTZ) and 42.27 mM (FOXO1:TFP).
It was further revealed that the PTZ hydrophobic binding pocket was lined mainly with residues Leu163, Leu168, Lys171, Trp189, Val194, and Pro195, and the hydroxylic Ser193 showed crucial interactions with the ligand (Figure 4). Similarly, TFP binding site was also hydrophobic with residues Leu183, Tyr187, Leu217, Arg225, Ser234, Ser235, and Trp237. Weak interactions with TFP were seen through Ser184, Ser218, Ser234, and Ser235. Besides, non-covalent hydrogen bonding was evident between TFP’s electrophilic F1, F2, and F3 and nucleophilic O of Arg214 (Figure 5). Molecular docking analysis was done to understand the binding efficiency of the selected drugs; PTZ was found to be the better FOXO1 inhibitor as it displayed a higher negative binding energy as compared to TFP, hence, it promises to be a more effective inhibitor.

3. Discussion

Generally, normal development requires only two copies of autosomal chromosomes; the presence of a third copy of chromosome (trisomy) is mostly lethal to the embryo. However, trisomy 13, 18, and 21 are the only cases where development can proceed to live birth. In the present study, the age of PS patients ranged from 1 day to 2 years, the majority (n = 19) died within a week, 8 within a month, 9 passed a month barring 1 surviving 2 years as an exception. Studies also showed survival with trisomy 13 being miserably limited with median life expectancy of 2.5 days. The overall observation reinforces other studies where 85% of PS patients could hardly survive beyond a month [2] and rarely survive beyond 10 years [8,9,10].
It is typical to have different types of abnormalities related to chromosome 13 manifested in various disorders. Apart from PS others include 13q deletion syndrome, propionic academia, retinoblastoma, Waardenburg Syndrome, Wilson’s Syndrome, Young–Madders Syndrome and also bladder and breast cancer. In the present case, all cases had confirmed trisomy 13. However, other researchers have reported full trisomy of chromosome 13 in 70–80% of cases, mosaicism in 10–20% and translocations involving chromosomes 13 in 5–10%, besides other types of chromosomal abnormalities in 5–10% of cases [15].
The frequency of CHD in patients was 61%, which falls in the range (56 to 86%) of frequency reported by other studies [3,15,77,78]. However, relatively low frequency has been reported by Rasmussen et al. (45.7%) and Pont et al. (34.8%) [4,79].
No plausible explanation is forthcoming as to how extra genetic material (trisomy 13) causes a plethora of abnormal features like abnormal cerebral functions, a small cranium, retardation, nonfunctional eyes, and heart imperfections. We made an attempt to identify important pathogenic genes such as EDNRB, ZIC2, ATP7B, GJB2, HTR2A located on chromosome 13 to associate these with diseases and pathways; however, none was alone capable of the symptoms of PS.
As for exploring the pathways, it is known that the EDNRB gene located at 13q22.3 codes for endothelin receptor type B protein, a GPCR located on the cell surface which functions via interaction with endothelins. It activates a phosphatidylinositol-calcium, the second messenger system transmitting information from outside the cell to inside. Its highest expression is in placental tissues. Mutations in this gene have been previously linked to the congenital genetic disorder, Hirschsprung disease, alternatively called Congenital Aganglionic Megacolon. It is a neural crest development disorder characterized by absence of enteric ganglia along a variable length of the intestine causing intestinal obstruction [80]. The Zic family member 2 (ZIC2) gene is present at 13q32.3 genomic region and encodes a type of zinc finger protein that functions as a transcriptional repressor and regulates both early and late stages of forebrain development. Mutations in ZIC2 gene, involving expansion of an alanine repeat at C-terminus, cause holoprosencephaly-5, a structural anomaly of the human brain [81,82,83]. It appears that a polyhistidine tract gene polymorphism is probably associated with increased risk of Holoprosencephaly. The defect appears to be due to changes in the organizer region leading to defective anterior notochord, further resulting in degradation of the prechordal plate. As a result shh signal cannot reach to the developing forebrain, vital for the formation of the two hemispheres [84]. ZIC2 has also been linked to neural tube defects [85] and heart defects [86].
The present endeavor extended search beyond chromosome 13 and identified genes such as NODAL, FPR1, AFP, AGO2, UROD, GATA4, GJA1, JAG1, CETED2, RYR2, NKX2-5, RARA, SIRT1, TBX5, AKT1, and PCK1 across genome with a view to exploring their role in PS. In doing so, two facts emerged clearly; one, the majority of PS patients had CHD and, two, all patients showed trisomy13. It thus appears that there could be a strong link between genes located on trisomy13 and heart disorders. Ingenuity pathway analysis of chr13 genes explored indicated hundreds of canonical pathways and many of them had FOXO1 as key molecules of such pathways. We applied a bioinformatics approach and searched scientific literature and identified pathogenic genes involved in CHD located on chr13 beside other chromosomes. It appears that genes of chr13 and other chromosomes might work together, either as transcription factor regulator or interacting partners. Nevertheless, FOXO1 is a strong transcription factor activating many genes, these being: FASLG, IGFBP1, SOD2, PPARGC1A, ADIPOQ, APOC3, OSTN, BCL2L11, CCND2, and CDKN1B. The protein–protein interaction study also showed key interacting partners like GATA4, NKX2-5, SIRT1, CITED2, NFATc1 and TBX5, which are actively implicated in heart disorders and thus partly responsible for PS.
It will not be out of place to mention GATA4, an interaction partner of FOXO1, a strong transcription factor regulating cardiac repair and remodeling. It plays an important role in cardiac development and differentiation as its abnormal expression leads to embryonic lethality [87,88,89]. Likewise, overexpression of NKX2-5 is reported as hypertrophic stimuli [90]. Interestingly, GATA4 and NKX2-5 act synergistically and regulate a myriad of cardiac genes [91,92]. Other studies showed that TBX5 is also an interaction partner of FOXO1, GATA4, and NKX2-5 and encodes transcription factors involved in the regulation of forelimb and heart development [93,94,95]. Thus, the role of GATA4, NKX2-5, and TBX5 is established in cardiogenesis; however, their role in regulating the heart septal formation is a matter of further investigation [45,96,97]. Sperling et al. are credited for reporting a direct role of CITED2 gene mutation in CHD epigenetic factor like methylation in the promoter region of CITED2 plays a vital role in heart disease [98,99]. The sirtuins, a family of enzymes, encoded by SIRT1–SIRT7, are highly expressed in the heart tissue and the vascular endothelium, and are pivotal regulators of lifespan and health. The SIRT1 executes its function by deacetylation of FOXO transcription factor and other key substrates; all closely linked to cardio vascular ailments. The SIRT1 inhibition is shown to be associated with septal and valvular heart defects, as well as vascular dysfunction [100,101,102].
One thing is evident clearly though multiple studies—there is a direct and indirect involvement of FOXO1 in heart disease [52,53,54,55]. Activated FOXO1 has a direct impact on cell survival via alteration in metabolism and turning on the cell death signaling cascade [103,104]. Overexpression of FOXO1 also causes autophagy in heart, leading to death [56,57]. A latest study had shown that knock down of FOXO1 and FOXO3 in the heart of Lmna−/− mice results in attenuation of apoptosis with a twice increase in the survival rate [18].
A bit of inhibiting expression of FOXO1 protein will further classify its important role in regulation. It is a monomeric nuclear protein and functions primarily as transcription factors by binding to a consensus DNA sequence of promoter region of downstream genes with a DNA-binding domain, 158–248 amino acid region [63,105,106,107]. The nuclear localization of FOXO1 is tightly regulated by the post translational modifications like acetylation, methylation, phosphorylation, and ubiquitination, or simply by its interaction with proteins like 14-3-3 [105]. Studies on these lines have identified a long list of FOXO1 inhibitors to be classified into two groups: one, drugs targeting nuclear transport of FOXO1 including leptomycin B, curcumin, psammaplysene A, phenothiazines/triflouperazine, calmodulin inhibitor/calmidazolium, intracellular Ca2+ chelator- BAPTA-AM l; and two, drugs targeting FOXO1 signaling pathway including epigallocatechin- 3-gallate, theaflavins, hyaluronan oligosaccharides, resveratrol, apigenin, luteolin, and psammaplysene A [64,65,67].
Phenothiazines and its derivatives (trifluoperazine) are chosen from FDA-approved drugs and binds directly with DBD of FOXO1 [70,71]. The molecular docking approach was applied to determine inhibition constant, predicting binding modes and defining the specific binding sites. The results showed that both the drugs can potentially inhibit FOXO1 protein. The drug PTZ mainly interacts with hydrophobic amino acids of the DNA-binding region (158–235) of the protein target. The TFP also binds inside the DNA-binding domain but the interacting residues are different from those in the case of PTZ binding. The contact or interaction surface value of docked ligand and protein is 421.011 Å2 for PTZ and 612.637 Å2 for TFP.
The present study is mainly based on a bioinformatics approach, so it can be associated with few limitations. It is proposed to undertake in silico finding to resolve the issue, and predictions are advised to be validated before final conclusion. Our finding suggests genetic engineering potentials in future.

4. Materials and Methods

4.1. Patients

A total of 37 cases including PS, dysmorphic features, multiple congenital anomalies, CHD and cleft palate were registered from Western region of Saudi Arabia through the King Abdulaziz University Hospital, Jeddah. The majority of individuals were newborns with multiple abnormalities including heart and neurological disorders. Peripheral blood (5–10 mL) was obtained after informed consent and a complete clinical and case history was recorded. Ethical approval for the study (G/017/27) was obtained from the King Abdulaziz University clinical research ethics board dated 09-06-2009 and the study strictly followed the standard Helsinki ethical guidelines during this research work.

4.2. Cytogenetics Study

A standard 72 h lymphocyte culture and GTG banding (G banding by Trypsin and Giemsa) were applied to peripheral blood in all patients. Microscopic examinations were done using 50 cells for each patient. In cases of suspected mosaicism, the number expanded to one hundred cells. Chromosomes were analyzed by semi-automatic Applied Imaging Karyotyper and karyotypes were described as per the International System for Human Cytogenetic Nomenclature (ISCN, 2016) [108].

4.3. Molecular Pathway and Gene Ontology Analysis

Biological significance of protein coding genes of chromosome 13 was interpreted by the Ingenuity Pathways Analysis software version 338830M (Ingenuity Systems, Redwood City, CA, USA). Significance of relationships between genes and functional frameworks was indicated by Fisher’s exact test p-values. The percentage and number of uploaded genes/molecules matching to genes of a canonical pathway were measured for significance, expressed as a score. The Molecule Activity Predictor was employed to predict the effects of a gene/molecule on other molecules of pathway.

4.4. Identification of Functionally Significant Interacting Proteins of FOXO1

Search Tool for the Retrieval of Interacting Genes/Proteins (STRING version 9.1, https://string-db.org/) was used to identify significant proteins interacting with FOXO1. The biological database and web resource of known and predicted protein interactions were utilized, derived from high-throughput experimental sources, text mining and co-expression [109,110,111].

4.5. Molecular Docking and Drug Design

A search was made for available three-dimensional structures of FOXO1 protein in the RCSB’s PDB database and retrieved five entries: 3CO6, 3CO7, 3COA, 4LG0, and 5DUI. All these structures were DNA-bound protein complexes. We proceeded with PDB code 3CO7:C. It corresponds to UniProtKB (Q12778, https://www.uniprot.org/help/uniprotkb) and the residues 1–154 were missing from the protein chain C.
Information was collected for structure of two selected compounds; Phenothiazine and trifluoperazine from ZINC database (available online: http://zinc.docking.org). It is a database of commercially available compounds [112]. We downloaded the mol2 file for ZINC ID 00028150 and 19418959 respectively. Structural analogs of TFP (31350265 and 39546119) were not considered for the present study.
Docking calculations for predicting binding modes and energies of two ligands phenothiazine (PTZ) and trifluoperazine (TFP) to protein (FOXO1) employed DockingServer [113], and AutoDock software for gasteiger partial charges addition to the ligand atoms, combining non-polar hydrogen atoms and defining rotatable bonds. Affinity grids were generated using the Autogrid tool [114]. AutoDock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking simulations were performed using Lamarckian genetic algorithm and the Solis & Wets local search algorithm (http://autodock.scripps.edu) [115]. Initial position, torsions, and orientation of the ligands were set randomly. All rotatable torsions were released during docking. All experimentation was resultant of 10 different runs set to finish after 250,000 energy evaluations. The population size was fixed to 150. Translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied during the search.

4.6. Statistical Analysis

χ2 analysis and Fisher’s exact test were used to compare the clinical features and proportion of chromosomal abnormalities in PS patients. The statistical analysis was carried out using MATLAB ver R2007a (The MathWorks, Natick, MA, USA).

5. Conclusions

Cytogenetic analysis of 37 Saudi PS patients showed full trisomy 13 without exception. Molecular interactions study of 308 protein coding genes located on chromosome 13 led to identification of significant genes such as: FOXO1, RB1, CCNA1, TFDP1, KL, IRS2, F10, F7 GJB6, GJA3, TUBA3C/TUBA3D, COL4A1, FLT1, KLF12, and ZIC2. The pathways (Estrogen-mediated S-phase entry, Extrinsic prothrombin activation pathway, Gap junction signaling, Docosahexaenoic acid signaling, VEGF signaling, Cell cycle: G1/S checkpoint regulation, IL-3 Signaling) were explored to find an association with PS. Molecular network analysis and protein–protein interaction study indicated FOXO1 as strong transcription factor which interacts with other key genes like GATA4, CITED and TBX5 located on different chromosomes but associated with lethal heart disorders in PS. Lethal genetic disorders are toughest to treat and many PS newborns die within a couple of days with severe complications without proper treatment. However, patients with a less severe condition have some chance of survival and could be diagnosed with an actual problem and treated (surgery or medicine) accordingly. The in silico molecular docking studies done separately indicated phenothiazine and trifluoperazine as efficient inhibitor for FOXO1 protein as potential drugs for septal defects patients and PS. Molecular docking indicated phenothiazine to be an efficient inhibitor for FOXO1 and a candidate for future drug target, especially in septal defects patients and PS cases. It is recommended to utilize the present outcome after validation in vitro and in vivo animal model approaches.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/19/11/3547/s1.

Author Contributions

Conceptualization, A.A., S.A., M.A.-Q., and S.K.; Methodology, S.K. and M.A.-Q.; Formal Analysis, S.K.; Investigation, M.A.-Q.; Writing—Original Draft Preparation, S.K. and M.A.-Q.; Writing—Review & Editing, A.A. and S.A.; Visualization, S.K.; Supervision, M.A.-Q. and A.A.; Project Administration, M.A.-Q. and A.A.; Funding Acquisition, S.A. and M.A.-Q. All authors read and approved the final manuscript.

Funding

This project was funded by the Deanship of Scientific Research (Project Award Number-G/017/27) King Abdulaziz University, Jeddah, Saudi Arabia.

Acknowledgments

The authors would like to thank Zeenat Mirza and Heba Abusamara for their significant contribution. Our special thanks to Waseem Ahmed for editing and improving the English language and Mohammad Amir Khan for help in typing. We would also like to thank lab-staffs for technical support and the Center of Excellence in Genomic Medicine Research for administrative support.

Conflicts of Interest

The authors declare no conflict of interests.

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Figure 1. Karyotyping result; (A) Normal Karyotype of Healthy female and (B) Trisomy 13 in all cases (male = 20 and female = 17) of Patau Syndrome. Red arrow shows trisomy 13.
Figure 1. Karyotyping result; (A) Normal Karyotype of Healthy female and (B) Trisomy 13 in all cases (male = 20 and female = 17) of Patau Syndrome. Red arrow shows trisomy 13.
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Figure 2. Estrogen-mediated S-phase Entry pathway derived from 308 protein coding genes of triosomy 13 (chromosome 13) using Ingenuity Pathway Analysis Tool.
Figure 2. Estrogen-mediated S-phase Entry pathway derived from 308 protein coding genes of triosomy 13 (chromosome 13) using Ingenuity Pathway Analysis Tool.
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Figure 3. Protein–protein Interaction Partners (GATA4, NKX2-5, SIRT1, CITED, NFATc1, TBX5) of FOXO1.
Figure 3. Protein–protein Interaction Partners (GATA4, NKX2-5, SIRT1, CITED, NFATc1, TBX5) of FOXO1.
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Figure 4. Molecular docking of phenothiazine with FOXO1 protein. (A) Depicting the molecular structure of phenothiazine; (B) Structure visualization of FOXO1 protein bound with ligand PTZ. The interacting residues are labeled in the binding site. (C) 2D plot of phenothiazine of FOXO1 showing ligand–protein interaction profiled by AutoDock software of Docking Server. Leu163, Leu168, Lys171, Trp189, Val194, Pro195, and Ser193 residues of FOXO1 showed crucial interactions with the phenothiazine.
Figure 4. Molecular docking of phenothiazine with FOXO1 protein. (A) Depicting the molecular structure of phenothiazine; (B) Structure visualization of FOXO1 protein bound with ligand PTZ. The interacting residues are labeled in the binding site. (C) 2D plot of phenothiazine of FOXO1 showing ligand–protein interaction profiled by AutoDock software of Docking Server. Leu163, Leu168, Lys171, Trp189, Val194, Pro195, and Ser193 residues of FOXO1 showed crucial interactions with the phenothiazine.
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Figure 5. Molecular docking of trifluoperazine with FOXO1 protein. (A) Depicting the molecular structure of trifluoperazine; (B) Structure visualization of FOXO1 protein bound with ligand TFP. The binding site is shown and the interacting residues are labeled. (C) 2D plot of trifluoperazine of FOXO1 showing ligand–protein interaction profiled by AutoDock software of Docking Server. Leu183, Tyr187, Leu217, Arg225, Arg234, Ser184, Ser218, Ser234, Ser235 and Trp237 residues of FOXO1 showed crucial interactions with the trifluoperazine.
Figure 5. Molecular docking of trifluoperazine with FOXO1 protein. (A) Depicting the molecular structure of trifluoperazine; (B) Structure visualization of FOXO1 protein bound with ligand TFP. The binding site is shown and the interacting residues are labeled. (C) 2D plot of trifluoperazine of FOXO1 showing ligand–protein interaction profiled by AutoDock software of Docking Server. Leu183, Tyr187, Leu217, Arg225, Arg234, Ser184, Ser218, Ser234, Ser235 and Trp237 residues of FOXO1 showed crucial interactions with the trifluoperazine.
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Table
Table 1. Important pathogenic genes located on chromosome 13.
Table 1. Important pathogenic genes located on chromosome 13.
Gene SymbolGene NameCytobandAssociated DiseaseAssociated PathwaysParalog
ATP7BATPase Copper Transporting Beta13q14.3Wilson Disease, Menkes DiseaseCardiac conduction; Ion channel transport; Transmembrane transport of small moleculesATP7A
BRCA2Breast cancer 2, early onset13q13.1Fanconi Anemia, and Breast CancerDNA Damage and Role of BRCA1 and BRCA2 in DNA repair
CAB39LCalcium-binding protein 39-like13q14.2Acute Monocytic LeukemiaRET signaling and mTOR signaling pathwayCAB39
COL4A1Collagen Type IV Alpha 1 Chain13q34Coronary artery diseaseCollagen chain trimerization, Integrin Pathway, ERK Signaling.COL4A5
DZIP1DAZ interacting zinc finger protein 113q32.1Acrodermatitis Enteropathica, Zinc-Deficiency TypeHedgehog signaling and GPCR signaling.DZIP1L
EDNRBEndothelin receptor type B13q22.3Waardenburg SyndromeCalcium signaling pathway and Prostaglandin Synthesis and RegulationEDNRA
ESDS-formylglutathione hydrolase13q14.2Wilson Disease and LeukocoriaGlutathione metabolism
FOXO1Forkhead box O 113q14.11Rhabdomyosarcoma 2, Alveolar and RhabdomyosarcomaRET signaling; PI3K/AKT activation; Common Cytokine Receptor Gamma-Chain Family Signaling Pathways; AGE/RAGE pathwayFOXO3
FLT1Fms-related tyrosine kinase 113q12.3Anal Canal Squamous Cell Carcinoma and Eclampsiap70S6K Signaling and Focal AdhesionKDR
GAS6Growth Arrest Specific 613q34Sticky platelet Syndrome, Acute Maxillary Sinusitis, Mesangial Proliferative GlomerulonephritisApoptotic Pathways in Synovial Fibroblasts, GPCR Pathway, ERK SignalingPROS1
GJB2Gap junction protein, beta 2, 26 kDa (connexin 26)13q12.11Vohwinkel Syndrome and Bart–Pumphrey SyndromeDevelopment Slit-Robo signaling and Gap junction trafficking.GJB6.
GJB6Gap junction protein, beta 6 (connexin 30)13q12.11Ectodermal Dysplasia 2, Clouston Type and Deafness, Autosomal Dominant 3BGap junction trafficking; Vesicle-mediated transportGJB2
GPC5Glypican-513q31.3Simpson–Golabi–Behmel Syndrome and Tetralogy of FallotGlycosaminoglycan metabolismGPC3
HMGB1Box 5 Box 113q12.313q12.3 Microdeletion Syndrome, Adenosquamous Gallbladder CarcinomaActivated TLR4 signaling; Cytosolic sensors of pathogen-associated DNA; Innate Immune SystemHMGB2
HTR2A5-HT2A receptor13q14.2Schizophrenia; Major Depressive DisorderCalcium signaling pathway; Signaling by GPCRHTR2C
MIPEPMitochondrial intermediate peptidase13q12.12Combined Oxidative Phosphorylation Deficiency 31
PCCAPropionyl Coenzyme A carboxylase, alpha polypeptide13q32.3Propionicacidemia and PCCA-Related Propionic Acidemia.Metabolism and HIV Life Cycle.MCCC1
RB1Retinoblastoma 113q14.2Retinoblastoma and Small-Cell Cancer of the Lung, Somatic.Arrhythmogenic right ventricular cardiomyopathy (ARVC) and DNA DamageRBL2
RCBTB1RCC1 and BTB domain-containing protein 113q14.2Retinal Dystrophy with Or Without Extraocular Anomalies. RCBTB2
RGCCRegulator of cell cycle RGCC13q14.11Renal Fibrosis and Retinal CancerTP53 Regulates Transcription of Cell Cycle Genes
RNR1Encoding RNA, ribosomal 45S cluster 113p12Idiopathic Bilateral Vestibulopathy and Congenital CytomegalovirusViral mRNA Translation
SLITRK6SLIT and NTRK-like protein 613q31.1Deafness and Yopia and Autosomal Recessive Non-Syndromic Sensorineural Deafness SLITRK5
SOX21Transcription factor SOX-2113q32.1Mesodermal Commitment Pathway and ERK Signaling.Mesodermal Commitment Pathway; ERK SignalingSOX14
STARD13StAR-Related Lipid Transfer Domain Containing 1313q13Hepatocellular Carcinoma, Arteriovenous Malformations of the Brain, Fibrosarcoma of Bonep75 NTR receptor-mediated signaling, Signaling by GPCR, Signaling by Rho GTPasesSTARD8
TPT1Translationally controlled tumor protein (TCTP)13q14.13Urticaria and AsthmaDNA Damage and Cytoskeletal Signaling
TRPC4Transient Receptor Potential Cation Channel Subfamily C Member 413q13.3Photosensitive EpilepsyDevelopmental Biology, Ion channel transport, Netrin-1 signalingTRPC5
TSC22D1TSC22 domain family protein 113q14.11Salivary Gland Cancer and Brain SarcomaDevelopment TGF-beta receptor signaling and Ectoderm DifferentiationTSC22D2
TUBA3CTubulin Alpha 3C13q12.11Clouston Syndrome, nonsyndromic Deafness, Kabuki Syndrome 1Development Slit-Robo signaling, Cooperation of Prefoldin and TriC/CCT in actin and tubulin foldingTUNA3D
XPO4Exportin-413q12.11Conjunctival Degeneration and PingueculaeIF5A regulation in response to inhibition of the nuclear export system and Ran Pathway
ZIC2Zic Family Member 213q32.3Holoprosencephaly 5 and Zic2-Related HoloprosencephalyMesodermal Commitment PathwayZIC1
ZMYM2Zinc finger MYM-type protein 213q12.11Lymphoblastic Lymphoma and 8P11 Myeloproliferative SyndromeHIV Life Cycle and FGFR1 mutant receptor activationZMYM3
Table
Table 2. Top canonical pathways determined by Ingenuity pathway analysis tools based on protein coding genes located on chromosome 13.
Table 2. Top canonical pathways determined by Ingenuity pathway analysis tools based on protein coding genes located on chromosome 13.
Canonical Pathways−log (p Value)RatioMolecules
Estrogen-mediated S-phase Entry2.060.115RB1, CCNA1, TFDP1
Cancer Signaling1.690.052RB1, FOXO1, TFDP1, KL, IRS2, CDK8, SMAD9, TFDP1, ARHGEF7
Extrinsic Prothrombin Activation Pathway1.560.125F10, F7
Role of p14/p19ARF in Tumor Suppression1.50.071RB1, KL, IRS2
Gap Junction Signaling1.410.036GJB6, KL, GJA3, TUBA3C/TUBA3D, IRS2, GJB2, HTR2A
Docosahexaenoic Acid (DHA) Signaling1.270.057FOXO1, KL, IRS2
Aldosterone Signaling in Epithelial Cells1.240.035SACS, KL, HSPH1, DNAJC3, IRS2, DNAJC15
FGF Signaling1.20.044KL, FGF9, FGF14, IRS2
GP6 Signaling Pathway1.180.038COL4A1, KL, IRS2, COL4A2, KLF12
Adipogenesis pathway1.170.037RB1, SAP18, SMAD9, FOXO1, KLF5
VEGF Signaling1.080.040FOXO1, FLT1, KL, IRS2
Cell Cycle: G1/S Checkpoint Regulation1.040.046RB1, FOXO1, TFDP1
ErbB2-ErbB3 Signaling0.9940.044FOXO1, KL, IRS2
Nitric Oxide Signaling in the Cardiovascular System0.9880.037FLT1, KL, SLC7A1, IRS2
Coagulation System0.9480.057F10, F7
Angiopoietin Signaling0.8750.039FOXO1, KL, IRS2
Role of NANOG in Mammalian Embryonic Stem Cell Pluripotency0.8660.0333SMAD9, KL, CDX2, IRS2
IL-3 Signaling0.8050.036FOXO1, KL, IRS2
Actin Cytoskeleton Signaling0.8010.027KL, FGF9, DIAPH3, ARHGEF7, FGF14, IRS2
14-3-3-mediated Signaling0.7780.030FOXO1, KL, TUBA3C/TUBA3D, IRS2
IL-7 Signaling Pathway0.7740.034FOXO1, KL, IRS2
HMGB1 Signaling0.770.030HMGB1, KL, IL17D, IRS2
NF-κB Signaling0.7690.028TNFSF11, FLT1, KL, IRS2, TNFSF13B
Table
Table 3. Key genes strongly associated with the survival of PS patient.
Table 3. Key genes strongly associated with the survival of PS patient.
Gene SymbolGene NameCytobandAssociated DiseaseAssociated PathwaysParalog
NODALNodal Growth Differentiation Factor10q22Visceral Heterotaxy 5 (HTX5) and Nodal-Related Visceral HeterotaxyMesodermal Commitment Pathway and Signaling pathways regulating pluripotency of stem cellsGDF3
FPR1Formyl Peptide Receptor 119q13.41Susceptibility to Localized Juvenile Periodontitis and Periodontitis 1, JuvenileSignaling by GPCR and Peptide ligand-binding receptorsFPR2
AFPAlpha Fetoprotein4q13.3Alpha-Fetoprotein Deficiency and Hereditary Persistence of Alpha-FetoproteinGlucocorticoid receptor regulatory network and Embryonic and Induced Pluripotent Stem Cell Differentiation Pathways and Lineage-specific MarkersALB
AGO2Argonaute RISC Catalytic Component 28q24.3Chromosome 18P Deletion Syndrome and Gum CancerRET signaling and Translational Control.AGO1
URODUroporphyrinogen Decarboxylase1p34.1Porphyria Cutanea Tarda and Urod-Related PorphyriasMetabolism and Porphyrin and chlorophyll metabolism
GATA4GATA Binding Protein 48p23.1Testicular Anomalies with or without Congenital Heart Disease and Atrial Septal Defect 2Response to elevated platelet cytosolic Ca2+ and Human Embryonic Stem Cell PluripotencyGATA6
GATA6GATA Binding Protein 618q11.2Pancreatic Agenesis and Congenital Heart Defects and Atrioventricular Septal Defect 5Mesodermal Commitment Pathway and Response to elevated platelet cytosolic Ca2+GATA4
GJA1Gap Junction Protein Alpha 16q22.31Oculodentodigital Dysplasia and Syndactyly, Type IiiDevelopment Slit-Robo signaling and Arrhythmogenic right ventricular cardiomyopathy GJA3
JAG1Jagged 120p12.2Alagille Syndrome 1 and Tetralogy of FallotSignaling by NOTCH1 and NOTCH2 Activation and Transmission of Signal to the NucleusJAG2
CITED2Cbp/P300 Interacting Transactivator with Glu/Asp Rich Carboxy-Terminal Domain26q24.1Atrial Septal Defect 8 and Ventricular Septal Defect 2Cellular Senescence (REACTOME) and Transcriptional regulation by the AP-2 (TFAP2) family of transcription factorsCITED1
RYR2Ryanodine Receptor 21q43Ventricular Tachycardia, Catecholaminergic Polymorphic, 1 and Arrhythmogenic Right Ventricular Dysplasia 2Calcium signaling pathway and Arrhythmogenic right ventricular cardiomyopathy RYR3
NKX2-5NK2 Homeobox 55q35.1Atrial Septal Defect 7, With or Without Av Conduction Defects and Tetralogy of FallotHuman Embryonic Stem Cell Pluripotency and NFAT and Cardiac HypertrophyNKX2-3
RARARetinoic Acid Receptor Alpha17q21.2Leukemia, Acute Promyelocytic, Somatic and Myeloid LeukemiaNuclear Receptors in Lipid Metabolism and Toxicity and Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3.RARB
CXCL12C-X-C Motif Chemokine Ligand 1210q11.21HIV-1 and AIDS Dementia Complexp70S6K Signaling and Akt Signaling
SIRT1Sirtuin 110q21.3Xeroderma Pigmentosum, Group D and Ovarian Endodermal Sinus TumorLongevity regulating pathway and E2F transcription factor networkSIRT4
TBX5T-Box 512q24.21Holt–Oram Syndrome and Aortic Valve Disease 2Human Embryonic Stem Cell Pluripotency and Cardiac conduction.TBX4
AKT1AKT Serine/Threonine Kinase 114q32.33Cowden Syndrome 6 and Proteus Syndrome, SomaticTranscription Androgen Receptor nuclear signaling and E-cadherin signaling in keratinocytesAKT3
CDKN2ACyclin Dependent Kinase Inhibitor 2A9p21.3Pancreatic Cancer/Melanoma Syndrome and Melanoma and Neural System Tumor SyndromeDNA Damage and Bladder cancerCDKN2B
PCK1Phosphoenolpyruvate Carboxykinase 120q13.31Pepck 1 Deficiency and Phosphoenolpyruvate Carboxykinase-1, Cytosolic, DeficiencyAbacavir transport and metabolism and Citrate cycle (TCA cycle)PCK2

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Abuzenadah, A.; Alsaedi, S.; Karim, S.; Al-Qahtani, M. Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies. Int. J. Mol. Sci. 2018, 19, 3547. https://doi.org/10.3390/ijms19113547

AMA Style

Abuzenadah A, Alsaedi S, Karim S, Al-Qahtani M. Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies. International Journal of Molecular Sciences. 2018; 19(11):3547. https://doi.org/10.3390/ijms19113547

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Abuzenadah, Adel, Saad Alsaedi, Sajjad Karim, and Mohammed Al-Qahtani. 2018. "Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies" International Journal of Molecular Sciences 19, no. 11: 3547. https://doi.org/10.3390/ijms19113547

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