1.Extraction of mitochondrial DNA from tooth dentin: application of two techniques
Ahmad Azlina a,b* ; Berahim Zurairah a ; Sidek Mohamad Ros b ; Mokhtar Khairani Idah a ; Samsudin Abdul Rani c
Archives of Orofacial Sciences 2011;6(1):9-14
Mitochondrial DNA (mtDNA) is a hereditary material
located in mitochondria and is normally maternally inherited.
Mutational analysis performed on mtDNA proved that the
mutations are closely related with a number of genetic
illnesses, besides being exploitable for forensic identification.
Those findings imply the importance of mtDNA in the scientific
field. MtDNA can be found in abundance in tooth dentin where
it is kept protected by the enamel, the hardest outer part of the
tooth. In this study, two techniques of mtDNA extraction were
compared to determine the efficacy between the two
techniques. Teeth used for the study was collected from Dental
Clinic, Hospital Universiti Sains Malaysia. After the removal of
tooth from the tooth socket of the patient, the tooth was kept at
-20C until use. Later, pulp tissue and enamel was excised
using dental bur and only the root dentin was utilized for the
isolation of mtDNA by crushing it mechanically into powdered
form. MtDNA was extracted using the two published methods,
Pfeifer and Budowle and then subjected to spectrophotometry
DNA quantification and purity, Polymerase chain reaction
(PCR) amplification of hypervariable-two region of mtDNA,
followed by DNA sequencing to analyze the reliability of the
extraction techniques. In conclusion, both techniques proved to
be efficient and capable for the extraction of mtDNA from tooth
dentin.
2.Cytogenomic Profiling of Chronic Lymphocytic Leukaemia Patients Using DNA Microarray
Wan Norizzati Wan Mohamad Zamri ; Nazihah Mohd Yunus ; Ahmad Aizat Abdul Aziz ; Mohamad Ros Sidek ; Noratifah Mohd. Adam ; Sarina Sulong
Malaysian Journal of Medicine and Health Sciences 2023;19(No.3):160-170
Introduction: Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the Western world. The
clinical presentation varies greatly, from very indolent cases to those with aggressive and fast advancing disease.
This variation has significant implications for clinical approaches, therapeutic tactics, and, ultimately, survival durations from diagnosis. Acquired chromosomal aberrations play a key role in CLL aetiology. Due to difficulty to obtain
abnormal metaphases for analysis, few methods such as fluorescence in-situ hybridization (FISH) and multiplex
ligation-dependent probe assay (MLPA) were employed to detect chromosomal aberration however the methods are
limited to specific locus only. Thus, this study is aimed to detect the chromosomal aberrations using DNA microarray platform. Methods: In this retrospective study, DNA archive obtained from 7 CLL patients which collected at
diagnosis and subjected to Affymetrix CytoScan® 750K single nucleotide polymorphism (SNP) array following the
manufacture procedure. The raw data obtained were analysed using the Chromosome Analysis Suite (ChAS) software (Affymetrix) using annotations of genome version GRCh38 (hg38). Result: Out of 7 patients, 4 of them showing
deletion of 13q while 3 of them showing deletion of 14q in various region . Some of the deleted loci were too small
(0.42-0.6Mb) to be detected by conventional cytogenetic analysis (CCA). There was also the presence of additional
chromosomal aberrations that could be missed by CCA, FISH, or MLPA due to cryptic deletion or duplication that
was as small as 0.4MB in size. Conclusion: The present study showed that low resolution chromosomal aberration
was able to be detected using DNA microarray platform in comparison to CCA, FISH and MLPA.
3.Application of HRM Analysis in Detection of PDGFRA Exon 10 Polymorphism in CML Patients with Imatinib Resistance
Nur Sabrina Abd Rashid ; Sarina Sulong ; Azlan Husin ; Rosline Hassan ; Mohamad Ros Sidek ; Nazihah Mohd Yunus
Malaysian Journal of Medicine and Health Sciences 2022;18(No.5):130-137
Introduction: Imatinib mesylate has been widely used as a standard treatment for chronic myeloid leukemia (CML).
It acts as a selective competitive inhibitor of the BCR-ABL tyrosine kinase. Despite the excellent efficacy on CML
treatment, some patients developed resistance to the treatment. Mutation in the PDGFRA may be one of the factors
involved in the mechanism of resistance that affects the response to imatinib. The mutational status of PDGFRA is
highly relevant for prognosis and treatment prediction in CML patients. Thus, this study is intended to establish and
validate a High Resolution Melting (HRM) analysis for PDGFRA exon 10 c.1432 T>C polymorphism in CML patients.
Methods: High resolution melting (HRM) analysis was used to identify the c.1432 T > C polymorphism in PDGFRA
exon 10 (n =86; response = 43; resistance = 43). The results from HRM analysis were compared and validated with
Sanger sequencing. The association between the polymorphism and treatment response was assessed by statistical
analysis using binomial logistic regression analysis. Results: HRM analyses showed two different melt curves. One
curve followed the shape of the reference, homozygous wild type (TT) and the other curve showed a different melting profile than the reference with the TC genotype (heterozygous variant). The results revealed that heterozygous
variant (TC) genotype showed a high risk of acquiring resistance with an OR of 3.795; 95% CI: 1.502-9.591, with
a statistically significant association, p = 0.005. HRM analysis also showed 100% sensitivity and specificity in the
detection of PDGFRA exon 10. Conclusion: The HRM analysis of PDGFRA exon 10 c.1432 T>C was successfully
established. The exon 10 c.1432 T>C polymorphism shows a higher risk for the development of resistance toward
imatinib treatment.