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Sequencing analysis of exons 5 and 6 in RUNX2 in non-syndromic patients with supernumerary tooth in Kelantan, Malaysia

Saharudin S, Sanusi SY, Ponnuraj KT

Clin Oral Investig, 2022 Feb;26(2):1261-1268.
PMID: 34453594 DOI: 10.1007/s00784-021-04098-x

OBJECTIVE: The aim of this study is to do a sequencing analysis of RUNX2 in non-syndromic patients with supernumerary tooth.
MATERIALS AND METHODS: Fifty-three patients with supernumerary tooth were identified retrospectively from 1,275 radiographic reviews who attended the Hospital Universiti Sains Malaysia (USM) Dental Clinic. Informed consent was obtained from the patients prior to the study. Blood samples were collected from 41 patients and DNA extractions were performed out of which 10 samples were chosen randomly for PCR amplification using designated primers for RUNX2 followed by DNA sequencing analysis.
RESULTS: This study involved 28 male patients (68.3%) and 13 female patients (31.7%) with a gender ratio of 2.2:1 and mean age of 15.9 ± 6.2 years. DNA extraction yielded ~ 40 ng/μl of concentrated DNA, and each DNA sample had more than 1500 bp of DNA length. The purity ranged between 1.8 and 2.0. DNA sequencing analysis did not reveal any mutations in exons 5 and 6 of RUNX2.
CONCLUSION: This study did not reveal any mutations in exons 5 and 6 of RUNX2 in non-syndromic patients with supernumerary tooth.
CLINICAL RELEVANCE: Analysis of mutations in RUNX2 is important to enhance the understanding of tooth development in humans.
Genotoxicity evaluation of dental restoration nanocomposite using comet assay and chromosome aberration test

Musa M, Ponnuraj KT, Mohamad D, Rahman IA

Nanotechnology, 2013 Jan 11;24(1):015105.
PMID: 23221152 DOI: 10.1088/0957-4484/24/1/015105

Nanocomposite is used as a dental filling to restore the affected tooth, especially in dental caries. The dental nanocomposite (KelFil) for tooth restoration used in this study was produced by the School of Dental Sciences, Universiti Sains Malaysia, Malaysia and is incorporated with monodispersed, spherical nanosilica fillers. The aim of the study was to determine the genotoxic effect of KelFil using in vitro genotoxicity tests. The cytotoxicity and genotoxicity of KelFil was evaluated using MTT assay, comet assay and chromosome aberration tests with or without the addition of a metabolic activation system (S9 mix), using the human lung fibroblast cell line (MRC-5). Concurrent negative and positive controls were included. In the comet assay, no comet formation was found in the KelFil groups. There was a significant difference in tail moment between KelFil groups and positive control (p < 0.05). Similarly, no significant aberrations in chromosomes were noticed in KelFil groups. The mitotic indices of treatment groups and negative control were significantly different from positive controls. Hence, it can be concluded that the locally produced dental restoration nanocomposite (KelFil) is non-genotoxic under the present test conditions.
Expression of Odontogenic and Osteogenic Markers in DPSCs and SHED: A Review

Ching HS, Luddin N, Rahman IA, Ponnuraj KT

Curr Stem Cell Res Ther, 2017;12(1):71-79.
PMID: 27527527

The odontogenic and osteogenic potential of dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous tooth (SHED) have been shown clearly by various in vitro and in vivo studies. The findings are promising and demonstrated that dental tissue engineering can give a new hope to the individuals suffering from tooth loss and dental diseases. The evaluation of odontogenic and osteogenic differentiation of DPSCs and SHED is commonly carried out by an illustration of the expression of varied related markers. In this review, few commonly used markers such as alkaline phosphatase (ALP), collagen type 1 (Col I), dentin matrix acid phosphoprotein 1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), osteocalcin (OCN), and osteopontin (OPN). DSPP, DMP1, and MEPE (odontogenic markers), which play an important role during early odontoblastic differentiation and late dentin mineralization, have been highlighted. Osteoblastic proliferation and early/late osteoblastic differentiation can be assessed by estimating the expression of Col I, ALP, OCN and OPN. Despite that, till date, there is no marker which could demonstrate for certain, the differentiation of human DPSCs and SHED towards the odontogenic and osteogenic lineage. This review suggests that SHED are noticeably different from DPSCs and exhibited higher capacity for osteogenic differentiation compared to DPSCs. On the other hand, different expression levels are shown by SHED and DPSCs with regards to the osteoblast markers for osteoblastic differentiation, where, SHED expressed higher levels of ALP, Col I and OCN compared to DPSCs.
Fulltext Molecular Detection of Alpha Thalassemia: A Review of Prevalent Techniques

Vijian D, Wan Ab Rahman WS, Ponnuraj KT, Zulkafli Z, Mohd Noor NH

Medeni Med J, 2021;36(3):257-269.
PMID: 34915685 DOI: 10.5222/MMJ.2021.14603

Alpha thalassemia (α-thalassemia) is an autosomal recessive disorder due to the reduction or absence of α globin chain production. Laboratory diagnosis of α-thalassemia requires molecular analysis for the confirmatory diagnosis. A screening test, comprising complete blood count, blood smear and hemoglobin quantification by high performance liquid chromatography and capillary electrophoresis, may not possibly detect all the thalassemia diseases. This review focused on the molecular techniques used to detect α-thalassemia, and the advantages and disadvantages of each technique were highlighted. Multiplex gap-polymerase chain reaction, single-tube multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, and loop-mediated isothermal amplification were used to detect common deletion of α-thalassemia. Furthermore, the reverse dot blot analysis and a single tube multiplex polymerase chain reaction could detect non-deletion mutation of the α-globin gene. Sanger sequencing is widely used to detect non-deletion mutations of α-thalassemia. Recently, next-generation sequencing was introduced in the diagnosis of both deletion and point mutations of α-thalassemia. Despite the advantages and disadvantages of different techniques, the routine method employed in the laboratory should be based on the facility, expertise, available equipment, and economic conditions.
Fulltext Mutagenicity and DNA damage assessment of locally produced nano-hydroxyapatite-silica-glass ionomer cement on human periodontal ligament fibroblasts using Ames and Comet assays

Niazi FH, Luddin N, Niazy A, Mohamad S, Harun MH, Noushad M, et al.

Saudi Dent J, 2024 Nov;36(11):1432-1437.
PMID: 39619708 DOI: 10.1016/j.sdentj.2024.08.009

AIM: This research assessed the mutagenicity and DNA damage of a novel type of nano-hydroxyapatite-silica glass ionomer cement (nano-HA-SiO2-GIC) and a conventional GIC (cGIC) using Ames and Comet assays.
METHODS: Cell viability was tested on human periodontal ligament fibroblasts (HPLFs) using 3.125 mg/ml, 6.25, 12.5, 25, 50, 100 and 200 mg/ml, on both types of GICs employing MTT assay. For the Comet assay, HPLFs were treated with IC50, IC25 and IC10 of test materials and the tail moments were measured. In the Ames test, four genotypic variants of strains of Salmonella typhimurium (TA100, TA98, TA1537 and TA1535) and a strain of Escherichia coli (WP2 uvrA) were employed. The material tested was extracted using sterile distilled water (0.2 g per ml) at 37 °C for 72 h. This was considered as 100 %, which was diluted to 50, 25, 12.5 and 6.25 % utilizing sterile distilled water. These five concentrations were incubated with the bacterial strains with and without metabolic activation (S9), along with appropriate positive controls. The number of revertant colonies was used to evaluate the outcome.
RESULTS: The highest cell viability (159.4 %) for nano-HA-SiO2-GIC was noticed at 3.125 mg/ml, while the lowest (24.26 %) was observed at 200 mg per ml. IC50, IC25 and IC10 values were 95.27, 51.4 and 20.1 mg/ml for cGIC, 106.9, 55.8 and 22.9 mg/ml for nano-HA-SiO2-GIC, respectively. The IC10 of both test materials showed no significant DNA damage compared to that of the negative control based on the Comet assay. The plate treated with nano-HA-SiO2-GIC showed less than double the average number of revertant colonies compared to that of negative control with regard to the Ames test.
CONCLUSIONS: It can be concluded that nano-HA-SiO2-GIC is non-mutagenic based on the Ames test and did not cause DNA damage at the lowest concentration of IC10 based on the Comet assay.
Fulltext Gene Mutation Spectrum among Alpha-Thalassaemia Patients in Northeast Peninsular Malaysia

Vijian D, Wan Ab Rahman WS, Ponnuraj KT, Zulkafli Z, Bahar R, Yasin N, et al.

Diagnostics (Basel), 2023 Feb 27;13(5).
PMID: 36900038 DOI: 10.3390/diagnostics13050894

(1) Background: Alpha (α)-thalassaemia is a genetic disorder that affects 5% of the world population. Deletional or nondeletional mutations of one or both HBA1 and HBA2 on chromosome 16 will result in reduced production of α-globin chains, a component of haemoglobin (Hb) that is required for the formation of red blood cells (RBCs). This study aimed to determine the prevalence, haematological and molecular characterisations of α-thalassaemia. (2) Method: The parameters were based on full blood count, high-performance liquid chromatography and capillary electrophoresis. The molecular analysis involved gap-polymerase chain reaction (PCR), multiplex amplification refractory mutation system-PCR, multiplex ligation-dependent probe amplification and Sanger sequencing. (3) Results: With a total cohort of 131 patients, the prevalence of α-thalassaemia was 48.9%, leaving the remaining 51.1% with potentially undetected α gene mutations. The following genotypes were detected: -α3.7/αα (15.4%), -α4.2/αα (3.7%), --SEA/αα (7.4%), αCSα/αα (10.3%), αAdanaα/αα (0.7%), αQuong Szeα/αα (1.5%), -α3.7/-α3.7 (0.7%), αCSα/αCSα (0.7%), -α4.2/αCSα (0.7%), -SEA/αCSα (1.5%), -SEA/αQuong Szeα (0.7%), -α3.7/αAdanaα (0.7%), --SEA/-α3.7 (2.2%) and αCSα/αAdanaα (0.7%). Indicators such as Hb (p = 0.022), mean corpuscular volume (p = 0.009), mean corpuscular haemoglobin (p = 0.017), RBC (p = 0.038) and haematocrit (p = 0.058) showed significant changes among patients with deletional mutations, but not between patients with nondeletional mutations. (4) Conclusions: A wide range of haematological parameters was observed among patients, including those with the same genotype. Thus, a combination of molecular technologies and haematological parameters is necessary for the accurate detection of α-globin chain mutations.