In-silico identification of deleterious non-synonymous SNPs of TBX1 gene: Functional and structural impact towards 22q11.2DS.

The TBX1 gene plays a critical role in the development of 22q11.2 deletion syndrome (22q11.2DS), a complex genetic disorder associated with various phenotypic manifestations. In this study, we performed in-silico analysis to identify potentially deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) within the TBX1 gene and evaluate their functional and structural impact on 22q11.2DS. A comprehensive analysis pipeline involving multiple computational tools was employed to predict the pathogenicity of nsSNPs. This study assessed protein stability and explored potential alterations in protein-protein interactions. The results revealed the rs751339103(C>A), rs780800634(G>A), rs1936727304(T>C), rs1223320618(G>A), rs1248532217(T>C), rs1294927055 (C>T), rs1331240435 (A>G, rs1601289406 (A>C), rs1936726164 (G>A), and rs911796187(G>A) with a high-risk potential for affecting protein function and stability. These nsSNPs were further analyzed for their impact on post-translational modifications and structural characteristics, indicating their potential disruption of molecular pathways associated with TBX1 and its interacting partners. These findings provide a foundation for further experimental studies and elucidation of potential therapeutic targets and personalized treatment approaches for individuals affected by 22q11.2DS.

Current guidelines, challenges and future recommendations for regulation of stem cell research and therapy: a commentary.

In recent years, Malaysia has seen a surge in stem cell therapy for various medical conditions. However, the regulation of stem cell research and therapy in Malaysia faces several challenges such as the emergence of unregulated clinics and a lack of specific legislation. Some urgent measures, including enactment of specific laws, strengthened monitoring, as well as increased public awareness and education, are crucial. Therefore, stem cell therapy regulation requires concerted efforts by the policymakers, regulator bodies and healthcare professionals. This commentary discusses the current guidelines and challenges in Malaysian stem cell therapy regulation and proposes some future recommendations that could pave the way for responsible progress of stem cell research and therapy globally.

Functionalization of poly (lactic-co-glycolic acid) nano­calcium sulphate and fucoidan 3D scaffold using human bone marrow mesenchymal stromal cells for bone tissue engineering application.

This study aimed to functionalize a novel porous PLGA (Poly lactic-co-glycolic acid) composite scaffold in combination with nano­calcium sulphate (nCS) and/or fucoidan (FU) to induce osteogenic differentiation of human bone marrow stromal cells. The composite scaffolds (PLGA-nCS-FU, PLGA-nCS or PLGA-FU) were fabricated and subjected to characterization using Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), Scanning electron microscopy (SEM) and Energy Dispersive X-Ray (EDX). The biocompatibility and osteogenic induction potential of scaffolds on seeded human bone marrow derived mesenchymal stromal cells (hBMSCs) were studied using cell attachment and alamar blue cell viability and alkaline phosphatase (ALP), osteocalcin and osteogenic gene expression, respectively. The composition of different groups was reflected in FTIR, XRD and EDX. The SEM micrographs revealed a difference in the surface of the scaffold before and after FU addition. The confocal imaging and SEM micrographs confirmed the attachment of cells onto all three composite scaffolds. However, the AB assay indicated a significant increase (p < 0.05) in cell viability/proliferation seeded on PLGA-nCS-FU on day 21 and 28 as compared with other combinations. A 2-fold significant increase (p < 0.05) in ALP and OC secretion of seeded hBMSCs onto PLGA-nCS-FU was observed when compared with other combinations. A significant increase in RUNX2, OPN, COL-I and ALP genes were observed in the cells seeded on PLGA-nCS-FU on day 14 and 28 as compared with day 0. In conclusion, the incorporation of both Fucoidan and Nano­calcium sulphate with PLGA showed a promising improvement in the osteogenic potential of hBMSCs. Therefore, PLGA-nCS-FU could be the ideal candidate for subsequent pre-clinical studies to develop a successful bone substitute to repair critical bone defects.

Identification of NRAS Diagnostic Biomarkers and Drug Targets for Endometrial Cancer-An Integrated in Silico Approach.

The diagnosis of endometrial cancer involves sequential, invasive tests to assess the thickness of the endometrium by a transvaginal ultrasound scan. In 6−33% of cases, endometrial biopsy results in inadequate tissue for a conclusive pathological diagnosis and 6% of postmenopausal women with non-diagnostic specimens are later discovered to have severe endometrial lesions. Thus, identifying diagnostic biomarkers could offer a non-invasive diagnosis for community or home-based triage of symptomatic or asymptomatic women. Herein, this study identified high-risk pathogenic nsSNPs in the NRAS gene. The nsSNPs of NRAS were retrieved from the NCBI database. PROVEAN, SIFT, PolyPhen-2, SNPs&GO, PhD-SNP and PANTHER were used to predict the pathogenicity of the nsSNPs. Eleven nsSNPs were identified as "damaging", and further stability analysis using I-Mutant 2.0 and MutPred 2 indicated eight nsSNPs to cause decreased stability (DDG scores < −0.5). Post-translational modification and protein−protein interactions (PPI) analysis showed putative phosphorylation sites. The PPI network indicated a GFR-MAPK signalling pathway with higher node degrees that were further evaluated for drug targets. The P34L, G12C and Y64D showed significantly lower binding affinity towards GTP than wild-type. Furthermore, the Kaplan−Meier bioinformatics analyses indicated that the NRAS gene deregulation affected the overall survival rate of patients with endometrial cancer, leading to prognostic significance. Findings from this could be considered novel diagnostic and therapeutic markers.

Predicting Deleterious Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) of HRAS Gene and In Silico Evaluation of Their Structural and Functional Consequences towards Diagnosis and Prognosis of Cancer.

The Harvey rat sarcoma (HRAS) proto-oncogene belongs to the RAS family and is one of the pathogenic genes that cause cancer. Deleterious nsSNPs might have adverse consequences at the protein level. This study aimed to investigate deleterious nsSNPs in the HRAS gene in predicting structural alterations associated with mutants that disrupt normal protein-protein interactions. Functional and structural analysis was employed in analyzing the HRAS nsSNPs. Putative post-translational modification sites and the changes in protein-protein interactions, which included a variety of signal cascades, were also investigated. Five different bioinformatics tools predicted 33 nsSNPs as "pathogenic" or "harmful". Stability analysis predicted rs1554885139, rs770492627, rs1589792804, rs730880460, rs104894227, rs104894227, and rs121917759 as unstable. Protein-protein interaction analysis revealed that HRAS has a hub connecting three clusters consisting of 11 proteins, and changes in HRAS might cause signal cascades to dissociate. Furthermore, Kaplan-Meier bioinformatics analyses indicated that the HRAS gene deregulation affected the overall survival rate of patients with breast cancer, leading to prognostic significance. Thus, based on these analyses, our study suggests that the reported nsSNPs of HRAS may serve as potential targets for different proteomic studies, diagnoses, and therapeutic interventions focusing on cancer.

A Metal-Containing NP Approach to Treat Methicillin-Resistant Staphylococcus aureus (MRSA): Prospects and Challenges.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of pneumonia in humans, and it is associated with high morbidity and mortality rates, especially in immunocompromised patients. Its high rate of multidrug resistance led to an exploration of novel antimicrobials. Metal nanoparticles have shown potent antibacterial activity, thus instigating their application in MRSA. This review summarizes current insights of Metal-Containing NPs in treating MRSA. This review also provides an in-depth appraisal of opportunities and challenges in utilizing metal-NPs to treat MRSA.

Anti- and Pro-Oxidant Properties of Essential Oils against Antimicrobial Resistance.

The rapid evolution of antimicrobial resistance (AMR) has remained a major public health issue, reducing the efficacy of antibiotics and increasing the difficulty of treating infections. The discovery of novel antimicrobial agents is urgently needed to overcome the challenges created by AMR. Natural products such as plant extracts and essential oils (EOs) have been viewed as potential candidates to combat AMR due to their complex chemistry that carries inherent pro-oxidant and antioxidant properties. EOs and their constituents that hold pro-oxidant properties can induce oxidative stress by producing reactive oxygen species (ROS), leading to biological damage in target cells. In contrast, the antioxidant properties scavenge free radicals through offsetting ROS. Both pro-oxidant and antioxidant activities in EOs represent a promising strategy to tackle AMR. Thus, this review aimed to discuss how pro-oxidants and antioxidants in EOs may contribute to the mitigation of AMR and provided a detailed description of the challenges and limitations of utilizing them as a means to combat AMR.

miRNA in Ischemic Heart Disease and Its Potential as Biomarkers: A Comprehensive Review.

Ischemic heart disease (IHD) constitutes the leading global cause of mortality and morbidity. Although significant progress has been achieved in the diagnosis, treatment, and prognosis of IHD, more robust diagnostic biomarkers and therapeutic interventions are still needed to circumvent the increasing incidence of IHD. MicroRNAs (miRNAs) are critical regulators of cardiovascular function and are involved in various facets of cardiovascular biology. While the knowledge of the role of miRNAs in IHD as diagnostic biomarkers has improved, research emphasis on how miRNAs can be effectively used for diagnosis and prognosis of IHD is crucial. This review provides an overview of the biology, therapeutic and diagnostic potential, as well as the caveats of using miRNAs in IHD based on existing research.

Screening of 22q11.2DS Using Multiplex Ligation-Dependent Probe Amplification as an Alternative Diagnostic Method.

BACKGROUND: The 22q11.2 deletion syndrome (22q11.2DS) is the most common form of deletion disorder in humans. Low copy repeats flanking the 22q11.2 region confers a substrate for nonallelic homologous recombination (NAHR) events leading to rearrangements which have been reported to be associated with highly variable and expansive phenotypes. The 22q11.2DS is reported as the most common genetic cause of congenital heart defects (CHDs). METHODS: A total of 42 patients with congenital heart defects, as confirmed by echocardiography, were recruited. Genetic molecular analysis using a fluorescence in situ hybridization (FISH) technique was conducted as part of routine 22q11.2DS screening, followed by multiplex ligation-dependent probe amplification (MLPA), which serves as a confirmatory test. RESULTS: Two of the 42 CHD cases (4.76%) indicated the presence of 22q11.2DS, and interestingly, both cases have conotruncal heart defects. In terms of concordance of techniques used, MLPA is superior since it can detect deletions within the 22q11.2 locus and outside of the typically deleted region (TDR) as well as duplications. CONCLUSION: The incidence of 22q11.2DS among patients with CHD in the east coast of Malaysia is 0.047. MLPA is a scalable and affordable alternative molecular diagnostic method in the screening of 22q11.2DS and can be routinely applied for the diagnosis of deletion syndromes.

Mutations in the tail domain of MYH3 contributes to atrial septal defect.

Atrial septal defect (ASD) is one of the most common congenital heart defects diagnosed in children. Sarcomeric genes has been attributed to ASD and knockdown of MYH3 functionally homologues gene in chick models indicated abnormal atrial septal development. Here, we report for the first time, a case-control study investigating the role of MYH3 among non-syndromic ASD patients in contributing to septal development. Four amplicons which will amplifies the 40 kb MYH3 were designed and amplified using long range-PCR. The amplicons were then sequenced using indexed paired-end libraries on the MiSeq platform. The STREGA guidelines were applied for planning and reporting. The non-synonymous c. 3574G>A (p.Ala1192Thr) [p = 0.001, OR = 2.30 (1.36-3.87)] located within the tail domain indicated a highly conserved protein region. The mutant model of c. 3574G>A (p.Ala1192Thr) showed high root mean square deviation (RMSD) values compared to the wild model. To our knowledge, this is the first study to provide compelling evidence on the pathogenesis of MYH3 variants towards ASD hence, suggesting the crucial role of non-synonymous variants in the tail domain of MYH3 towards atrial septal development. It is hoped that this gene can be used as panel for diagnosis of ASD in future.

Screening of SMAD7 in Malay patients with ventricular septal defect.

Ventricular septal defect (VSD) is the most common form of cardiac malformations accounting approximately 20% of all congenital heart defects. SMAD7 is an inhibitory protein that antagonizes the signalling of TGF-ß family member and has been found in the development and function of mouse heart models. This study aims to screen and identify the polymorphisms of SMAD7 exonic regions in Malay population with VSD. Peripheral blood samples were collected and extracted from 30 clinically diagnosed VSD patients. PCR amplification was performed using 12 sets of designed primers encompassing seven exons of SMAD7. Re-sequencing was conducted to characterize the polymorphisms of SMAD7. Observed polymorphisms were then genotyped in 30 healthy individuals using both re-sequencing and allele-specific PCR techniques. A total of 10 variants were identified in the patient population located in the upstream (rs7236774), exonic (rs368427729, rs145686330, rs3764482, rs3809922, rs780863704 and rs3809923), intronic (rs3736242) and 3'UTR regions (rs375444823 and rs16950113). No significant difference of genotype and allele frequency was observed among these SNPs. Two synonymous variants (rs3809922 and rs3809923) were found in complete linkage disequilibrium (r2=1.0) with each other indicate a strong correlation of these SNPs. The identification of these SNPs provides a new perspective of the VSD causation.

A whole genome analyses of genetic variants in two Kelantan Malay individuals.

BACKGROUND: The sequencing of two members of the Royal Kelantan Malay family genomes will provide insights on the Kelantan Malay whole genome sequences. The two Kelantan Malay genomes were analyzed for the SNP markers associated with thalassemia and Helicobacter pylori infection. Helicobacter pylori infection was reported to be low prevalence in the north-east as compared to the west coast of the Peninsular Malaysia and beta-thalassemia was known to be one of the most common inherited and genetic disorder in Malaysia. RESULT: By combining SNP information from literatures, GWAS study and NCBI ClinVar, 18 unique SNPs were selected for further analysis. From these 18 SNPs, 10 SNPs came from previous study of Helicobacter pylori infection among Malay patients, 6 SNPs were from NCBI ClinVar and 2 SNPs from GWAS studies. The analysis reveals that both Royal Kelantan Malay genomes shared all the 10 SNPs identified by Maran (Single Nucleotide Polymorphims (SNPs) genotypic profiling of Malay patients with and without Helicobacter pylori infection in Kelantan, 2011) and one SNP from GWAS study. In addition, the analysis also reveals that both Royal Kelantan Malay genomes shared 3 SNP markers; HBG1 (rs1061234), HBB (rs1609812) and BCL11A (rs766432) where all three markers were associated with beta-thalassemia. CONCLUSIONS: Our findings suggest that the Royal Kelantan Malays carry the SNPs which are associated with protection to Helicobacter pylori infection. In addition they also carry SNPs which are associated with beta-thalassemia. These findings are in line with the findings by other researchers who conducted studies on thalassemia and Helicobacter pylori infection in the non-royal Malay population.

Gastric precancerous lesions are associated with gene variants in Helicobacter pylori-susceptible ethnic Malays.

AIM: To identify genes associated with gastric precancerous lesions in Helicobacter pylori (H. pylori)-susceptible ethnic Malays. METHODS: Twenty-three Malay subjects with H. pylori infection and gastric precancerous lesions identified during endoscopy were included as "cases". Thirty-seven Malay subjects who were H. pylori negative and had no precancerous lesions were included as "controls". Venous blood was collected for genotyping with Affymetrix 50K Xba1 kit. Genotypes with call rates < 90% for autosomal single nucleotide polymorphisms (SNPs) were excluded. For each precancerous lesion, associated SNPs were identified from Manhattan plots, and only SNPs with a χ² P value < 0.05 and Hardy Weinberg Equilibrium P value > 0.5 was considered as significant markers. RESULTS: Of the 23 H. pylori-positive subjects recruited, one sample was excluded from further analysis due to a low genotyping call rate. Of the 22 H. pylori-positive samples, atrophic gastritis only was present in 50.0%, complete intestinal metaplasia was present in 18.25%, both incomplete intestinal metaplasia and dysplasia was present in 22.7%, and dysplasia only was present in 9.1%. SNPs rs9315542 (UFM1 gene), rs6878265 (THBS4 gene), rs1042194 (CYP2C19 gene) and rs10505799 (MGST1 gene) were significantly associated with atrophic gastritis, complete intestinal metaplasia, incomplete metaplasia with foci of dysplasia and dysplasia, respectively. Allele frequencies in "cases" vs "controls" for rs9315542, rs6878265, rs1042194 and rs10505799 were 0.4 vs 0.06, 0.6 vs 0.01, 0.6 vs 0.01 and 0.5 vs 0.02, respectively. CONCLUSION: Genetic variants possibly related to gastric precancerous lesions in ethnic Malays susceptible to H. pylori infection were identified for testing in subsequent trials.

Deleted in Colorectal Cancer (DCC) gene polymorphism is associated with H. pylori infection among susceptible Malays from the north-eastern region of Peninsular Malaysia.

BACKGROUND/AIMS: Using genome-wide case-control association approach, the current study aimed to determine whether genetic polymorphism(s) is/are associated with H. pylori infection among ethnic Malays from the north-eastern region of Peninsular Malaysia, a region with an exceptionally low prevalence for H. pylori infection and gastric cancer. METHODOLOGY: Twenty-three Malay subjects positive for H. pylori confirmed with urease test and histology were enrolled as "cases" and 37 subjects negative for H. pylori were "controls". Both groups were matched for age and environmental risks. Extracted DNA samples (QIAGEN, Germany) from the venous blood of study subjects were genotyped using the Human Mapping 50k xbal array (Affymetrix, USA). High throughput downstream analyses were then used to determine the significant SNP(s) associated with H. pylori infection. RESULTS: Out of 20,361 SNPs filtered using the genotype association test, the top 1% (203) significant SNPs were selected for functional enrichment analysis. Of the 15 "enriched" SNPs, the rs10502974 which was located within the intronic region of Deleted in Colorectal Cancer (DCC) gene was the SNP most significantly associated with H. pylori infection (p=0.00549). CONCLUSIONS: Ethnic Malays is genetically susceptible to H. pylori infection and is possibly mediated through a genetic variation in the DCC gene.

Towards understanding the low prevalence of Helicobacter pylori in Malays: genetic variants among Helicobacter pylori-negative ethnic Malays in the north-eastern region of Peninsular Malaysia and Han Chinese and South Indians.

OBJECTIVES: To identify gene polymorphisms that differ between Malays, Han Chinese and South Indians, and to identify candidate genes for the investigation of their role in protecting Malays from Helicobacter pylori (H. pylori) infection. METHODS: Malay participants born and residing in Kelantan with a documented absence of H. pylori infection were studied. Venous blood was used for genotyping using the Affymetrix 50K Xba I kit. CEL files from 141 Han Chinese and 76 South Indians were analyzed to compare their allele frequency with that of the Malays using fixation index (FST ) calculation. The single nucleotide polymorphisms (SNPs) with the highest allele frequency (outliers) were then examined for their functional characteristics using F-SNP software and the Entrez Gene database. RESULTS: In all, 37 Malays were enrolled in the study; of whom 7 were excluded for low genotyping call rates. The average FST estimated from the genome-wide data were 0.038 (Malays in Kelantan vs the South Indians), 0.015 (Malays in Kelantan vs Han Chinese) and 0.066 (Han Chinese vs South Indians), respectively. The outlier gene variants present in Malays with functional characteristics were C7orf10 (FST 0.29988), TSTD2 (FST 0.43278), SMG7 (FST 0.29877) and XPA (FST 0.43393 and 0.43644). CONCLUSION: Genetic variants possibly related to protection against H. pylori infection in ethnic Malays from the north-eastern region of Peninsular Malaysia were identified for testing in subsequent trials among infected and uninfected Malays.