Ghazali, Norliana
(2022)
Prevalence of dental anomalies and
genetic aberrations of nonsyndromic
cleft-lip with or without
cleft palate patients with
hypodontia.
PhD thesis, Universiti Sains Malaysia.
Abstract
Non-syndromic cleft lip and or without cleft palate (NSCL/P) with dental
anomalies are a common craniofacial abnormality in humans and animals. NSCL/P is
reported to have a higher prevalence of dental anomalies compared to non-cleft
individuals. This study aims to determine the prevalence of dental anomalies and to
identify the genetic aberration among NSCL/P children using DNA microarray. A
cross-sectional study was carried out at Hospital USM with a clinical oral examination
followed by an orthopantomogram analysis among NSCL/P and non-cleft children
between the ages of 7 to 13 years old. Saliva was collected from the subjects for
genomic DNA extraction. Extracted genomic DNA from 61 NSCL/P and 20 non-cleft
were subjected to the CytoScan 750K Array to identify genetic aberrations such as
copy number variations (CNVs) and loss of heterozygosity (LOH). For CytoScan
750K Array, 250 ng of genomic DNA were digested with Nsp1, before being ligated
to an adapter. Polymerase chain reaction (PCR) using a single pair of primers that
recognised the adapter sequence was then performed. All four aliquots of PCR
products were then combined and purified with magnetic beads followed by
fragmentation with DNase 1 then subsequently end-labeled with biotin and hybridyzed
into the CytoScan arrays. Next, the array was stained using GeneChips Fluidics Station
450 before scanning the arrays. The data was then analysed manually using the Chromosome Analysis Suite (ChAS) software to examine the genome. Statistical data
analysis was performed using Mann-Whitney and Chi-Square test in the IBM SPSS
software to confirm the significant results. The results showed that the prevalence
(95% CI) of anomalies related to the number of teeth (67%:95% CI: 0.540,0.787) was
higher than the morphology (8%;95% CI: 0.027, 0.181) in the NSCL/P children. The
highest dental anomalies was hypodontia among NSCL/P and non-cleft children. Six
significant CNVs were identified including gains (12q14.3, 15q26.3, 1p36.32, and
1p36.33) and losses (3p14.2 and 4q13.2) in the NSCL/P with hypodontia patients
compared to the NSCL/P only. The genes encoded in these regions are LEMD3,
IGF1R, TP73, SKI, FHIT, and UGT2β15. For LOH, the most recurrent regions were
found at 1p33-1p32.3, 1q32.2-1q42.13, and 6p12.1-6p11.1 loci occurred in 13 (16%),
5 (6%) and 7 (9%) among NSCL/P and non-cleft with hypodontia children,
respectively. Validation analysis revealed a significant copy number gain in TP73,
SKI, IGF1R and LEMD3 and loss in FHIT and UGT2β15 in NSCL/P children with
hypodontia (p < 0.005). Microsatellite markers analysis found a significant association
between D1S197 (1p36.33-32.3) markers in NSCL/P with hypodontia. In conclusion,
the present study has successfully determined the prevalence of dental anomalies and
identified the genetic aberrations among NSCL/P. The current study shows a novel
finding of copy number loss at 3p14.2 and 4q13.2 that includes FHIT and UGT2β15
which may contribute to the formation of NSCL/P with hypodontia. These results have
an immerse prospect in the promising field of individualized preventive oral health
care.
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