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Table of Contents   
ORIGINAL RESEARCH  
Year : 2022  |  Volume : 33  |  Issue : 2  |  Page : 158-163
Use of pulp tissue of deciduous teeth for gender determination - A comparative molecular analysis study


1 Department of Paediatric and Preventive Dentistry, AB Shetty Memorial Institute of Dental Sciences, NITTE Deemed to be University, Mangalore, Karnataka, India
2 Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, NITTE Deemed to be University, Mangalore, Karnataka, India

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Date of Submission25-May-2021
Date of Decision07-May-2022
Date of Acceptance13-Jun-2022
Date of Web Publication13-Oct-2022
 

   Abstract 


Aim: The present study aimed to evaluate the effectiveness of pulp tissue collected from deciduous teeth for the determination of gender using polymerase chain reaction (PCR). Materials and Methods: 140 extracted deciduous teeth were selected. The control group comprised 20 teeth that were subjected to DNA analysis immediately. Whereas Group I and Group II consisted of 60 teeth which were stored in the open environment and salt water, respectively, for a period of 3, 9, and 15 months. DNA was isolated and quantified followed by the amplification of X and Y chromosomes by PCR and compared with the actual gender of the child. The data were analysed using the Shapiro-Wilk test, the independent sample t-test, paired t-test, and the Chi-square test. Result: The PCR analysis results of Group I showed a more correct interpretation of gender as compared to Group II on storage for a period of 15 months. The PCR analysis results of the Control group showed a 100% accuracy rate as compared to the samples in Groups I and II. Conclusions: Gender could be effectively determined from the samples evaluated immediately after extraction. But the period of storage and the method of storage conditions affected the quality of isolated DNA and thus decreased the ability of gender determination.

Keywords: Deciduous teeth, gender determination, polymerase chain reaction, pulp tissue, salt water

How to cite this article:
Manju R, Ravi M S. Use of pulp tissue of deciduous teeth for gender determination - A comparative molecular analysis study. Indian J Dent Res 2022;33:158-63

How to cite this URL:
Manju R, Ravi M S. Use of pulp tissue of deciduous teeth for gender determination - A comparative molecular analysis study. Indian J Dent Res [serial online] 2022 [cited 2022 Nov 29];33:158-63. Available from: https://www.ijdr.in/text.asp?2022/33/2/158/358452



   Introduction Top


Identification of a person by dental means is considered as a reliable method for gender identification when other methods fail because of the advanced decomposition or skeletonization of the body.[1] In cases of severe decomposition or incineration of the body, the post-mortem survival of the teeth has gained more attention in forensic investigations even after the soft and skeletal tissues are destroyed.[1]

Due to the lack of sufficiently developed sexual characteristics, gender determination from skeletal remains of children and preadolescents is difficult.[2],[3] In such cases, the unique characteristics of dental tissues play an important role in personal identification.

Deoxyribonucleic acid (DNA) obtained from the dental pulp tissue, encased in a hard tissue casing of enamel and dentin, is an excellent source of DNA.[4]

It has been shown that the extraction of DNA from the pulp tissue of teeth is possible with sufficient quality and quantity to conduct a Polymerase chain reaction (PCR)-based analysis in cases of the exposure of the body to various environmental conditions like burial, mutilation, explosion, or incineration. It has also been reported that the pulp tissue produced the strongest PCR amplification signals while dentin and cementum signals were very similar to each other.[5]

The availability of DNA for forensic identification depends largely on the factors such as the type, moisture content, pH, temperature, and the microbial ecosystem of the environment in which the body is exposed which also play a significant role in the rate of dehydration of the pulpal tissue.[6]

Since children are highly vulnerable to abuse or maltreatment, it is possible that in a real forensic situation the body of the deceased can be either left on the ground or thrown into the water bodies in a hurry or buried in the earth which might remain there for months or years together and the availability of a deciduous tooth may be the only evidence available to identify the victim.

Considering these factors, the present study was undertaken to find out the efficacy of gender determination in children from the pulp tissue of exfoliated deciduous teeth stored exposed to dry and wet environmental conditions for several months by PCR technique.


   Materials and Methods Top


After obtaining informed consent from the parents, a total of 140 non-carious primary teeth showing pre-shedding mobility in children between the age group of 8 to 13 years were extracted and selected for the study. All the collected teeth had the roots completely resorbed. The study was carried out from the 1st of January 2020 to the 31st of May 2021. Clearance from Institutional Ethics Committee (Ref: NU/CEC/Ph. D-17/2014) was obtained.

Groups I and II teeth were divided into three subgroups of 20 teeth each (A, B, and C) and stored in the open environment and salt water for a period of 3, 9, and 15 months, respectively. Coding and decoding of all samples were performed.

Immediately after the extraction, the teeth samples designated for both the groups were decontaminated by washing with sterile distilled water. Group I samples were stored in sterile-labelled bottles and Group II samples in bottles containing seawater for the decided duration of time before the collection of the pulp tissue. Highly aseptic precautionary measures were taken while handling the teeth samples to avoid cross-contamination of samples.

Since the roots of primary molar teeth samples were completely resorbed, the pulp tissue was collected from the pulp chamber. Primary anterior teeth were sectioned longitudinally, and pulp tissue was collected using a sterile disposable needle. In cases of posterior teeth, the pulp tissue was collected from the pulp chamber by a sharp spoon excavator without sectioning the teeth. Even after the storage period of 15 months duration, the pulp tissue could be collected from all teeth samples, though in a reduced quantity. The collected pulp tissue was stored in labelled bottles of Dulbecco's phosphate buffer saline (PBS) and stored at −20°C till the DNA extraction.

Isolation of DNA from the pulp tissue was carried out using the NucleoSpin Tissue kit (DSS Takara Bio India Pvt. Ltd.). The tissue sample was placed in a micro-centrifuge tube of 1.5 mL, and 180 μL of buffer T1 was added to 25 μL of proteinase K solution and vortexed. Care was taken that the sample was completely covered with the lysis solution. The samples were then incubated at 56°C until complete lysis was obtained. Samples were then vortexed vigorously and incubated at 70°C for 10 minutes, and 600 μL of buffer was added and centrifuged at 11,000 rpm for 1 minute.

The isolated DNA from the pulp tissue was then quantified using Bio Spectrometer to evaluate the amount of DNA in μg/mL obtained from the samples of groups I and II. The DNA sample concentration was then measured using the software and was checked at 260 nm/280 nm range and recorded. The purity of DNA was in the range of 1.8—1.9.

In the present study, two sets of oligonucleotide primers were used. Four heat stable Taq DNA polymerase was provided in the PCR buffer kit. The heating cycles of PCR were preheating at 95°C for 3 minutes and 35 heating cycles (94°C for 40 s, 55°C for 40 s, and 72°C for 40 s) using a thermocycler.

PCR Primers selected were:

Primers: Y chromosome (size 172 bp):

  • Y11: 5′-ATGATAGAAACGGAAATATG.
  • Y22: 5′-AGTAGAATGCAAAGGGCTC.


Primers: X chromosome (size 131 bp):

  • X1: 5′-AATCATCAAATGGAGATTTG.
  • X2: 5′-GTTCAGCTCTGTGAGTGAAA.


The PCR products were then electrophoresed in 2% agarose gel at 80 to 100 V for 1 hour, ethidium bromide staining was performed, and amplified bands of Y- and X-specific band sequences were examined using Gel DOC™ with Image Lab™ software (Bio-Rad). The gender of the subjects was considered to be male when both Y- and X-specific sequences were detected, but female when only X-specific sequences were detected [Figure 1] and [Figure 2].
Figure 1: Representative image showing PCR amplification of X and Y specific chromosomes in a male sample. Both X- and Y-specific DNA bands amplified. M-DNA ladder, NC-Negative control, bp-Base pairs

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Figure 2: Representative image showing PCR amplification of X-specific chromosome in a female sample. Only X-specific DNA bands amplified. M-DNA ladder, NC-Negative control, bp-Base pairs.

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Shapiro–Wilk test was used to analyse the normality of data. As the data did not follow normal distribution, the non-parametric tests were used to analyse the data. To check to mean differences among the groups the Kruskal—Wallis test was used. Posthoc analysis was done using Dunn's test. A Chi-square test was used to compare the outcome between the different time intervals. The results of DNA quantification and PCR analysis of each group were statistically analysed using SPSS (Statistical Package for Social Sciences) Version 24.0 (BM Corporation, Chicago, USA).


   Results Top


PCR analysis was possible in all the teeth samples in the control group and showed correct interpretation of gender in all samples.

Intra-group comparison of mean DNA volume obtained from the pulp tissue in Group I has shown that there was no statistically significant difference between Groups IA with IB and also between the Groups IB and IC [Table 1].
Table 1: Intragroup comparison of mean DNA volume (ng/μl) obtained from Group I

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The mean DNA volume obtained from Group IIA was significantly more than from Groups IB But there was no significant difference between the Groups IIB and IIC [Table 2].
Table 2: Intragroup comparison of mean DNA volume (ng/μL) obtained from Group II

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Intragroup comparison of the PCR analysis of Group I have shown that after 3 months of storage, 15% of teeth samples showed mismatch. After 9 and 15 months, the percentage of mismatch was 20% and 10% respectively, and the percentage of non-amplification was 10% and 25% respectively. There was no statistical difference between groups IA, IB and IC. [Table 3]
Table 3: Intragroup comparison of PCR analysis in Group I

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PCR analysis has shown that after 3 months of storage in salt water, 20% of teeth samples showed mismatch and 10% of samples shown non-amplification of DNA. After 9 and 15 months, the percentage of mismatch was 5% each. The percentage of non-amplification was 10% and 50% respectively. PCR analysis could not be done in 15% of samples after 9 months and in 10% of samples after 15 months. There was a statistically significant difference in the PCR results between Groups IIA and IIC and also between Groups IIB and IIC [Table 4].
Table 4: Intragroup comparison of PCR analysis in Group II

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Intergroup comparison of PCR analysis results of the control group and Group I has shown that there was no statistical difference between the groups, but control group samples showed statistically significant differences with Group IB as well as Group IC. Intergroup comparison of PCR analysis results of the control Group II teeth has shown that there was a statistically significant difference between the control group and Groups IIA [Table 5].
Table 5: Intergroup comparison of PCR analysis between Control group, Group I and Group II

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   Discussion Top


Since the deciduous teeth show much less sexual dimorphism as compared to permanent teeth, gender determination from the external morphological characteristics of deciduous teeth is difficult. Hence one has to solely depend on the DNA available from primary teeth for personal identification.[3],[7] Hence, in the present study, pulp tissue was used as a source of DNA to identify the gender by molecular analysis

In the present-day scenario, with the existing socioeconomic conditions, children are more vulnerable to exploitation and abuse. Another major public health problem is the increase in mortality due to drowning. A study carried out in Kerala had shown that children between the age groups of 5—9 and 10—12 years are at high risk of drowning.[8]

PCR has gained much popularity owing to its ability to determine the gender even after months or years of death utilizing the minute quantity of DNA.

Although there are several studies on DNA analysis from pulp tissue of permanent teeth, only limited studies have been carried out in the deciduous dentition. Hence the present study was carried out in the deciduous teeth stored in a dry open environment as well as in salt water (wet environment). The pulp tissue was collected from the pulp chambers of the teeth as all the samples used in the study had the roots completely resorbed. Sufficient amount of pulp tissue could be collected from the chamber even after storing the samples for 15 months both in wet and dry conditions.

The first step in the laboratory processing for the PCR analysis was the isolation of DNA which was performed using a NucleoSpin tissue kit followed by quantification of DNA by Bio spectrometer. PCR analysis was carried out in all the teeth samples by denaturation, annealing, and extension. In the present study, PCR was used to amplify specific alphoid centromeric repeat sequences. The alphoid (alpha) satellite family is the only repetitive DNA family likely to exhibit the properties of significant chromosome specificity and is located in peri-centromeric regions of all human chromosomes.[9] Hence, in the present study alphoid repeat primers were used for the accurate sex determination by the detection of X and Y chromosome-specific alphoid repeat sequences which was in agreement with another study in the literature.[10]

Highest quantity of DNA was obtained from teeth which were processed immediately after extraction (control Group) when compared with that of the study groups. Intragroup comparison of the mean DNA volume obtained from Group I have shown no statistical difference irrespective of the reduction in mean DNA volume on prolonged storage up to 15 months. Similar findings were reported by Jacqueline B. Duffy et al. (1990), Ionesiy A. G (1980), Raphael S. S (1976), and Suresh Vemuri et al. (2016) who stated that as the pulpal tissue dries, there is an arrest of the necrotic and/or putrefactive processes resulting in its prolonged stability and diagnostic ability.[11],[12],[13],[14],[15] It is also stated that the amount of DNA decreases as the number of days of storage is increased.[15]

The PCR analysis carried out immediately after extraction showed 100% accurate results which reduced to 85%, 70%, and 65%, respectively, as the period of storage extended from 3, 9, and 15 months. There was a reduction in the quantity along with the quality of DNA as the period of storage increased. This could be due to the putrefaction of the specimens as the time proceeded or due to local conditions.[3]

The PCR analysis results of teeth samples stored in salt water showed 70%, 70%, and 35% accurate results, respectively, as the period of storage extended from 3, 9, and 15 months. There was a statistically significant difference between the PCR results of the teeth samples stored in salt water for 3 months and 15 months as well as 9 months and 15 months. There was a greater degradation of DNA in salt water. This could be due to the chemical composition of aquatic systems as well as the dilution effect of water itself. Microbial growth and humidity in the aquatic ecosystems have been reported to degrade genetic material[16] or inhibit the Taq DNA polymerase enzyme (humic acid) which is a hindrance to PCR.[17]

The nonamplification of the samples in both groups could be attributed to the effect of dry and wet conditions on the quantity and quality of DNA. The insignificant amount of biological material for DNA extraction may result in absence of the target sequence in the fraction used for the PCR reaction or the same can be degraded, not allowing DNA amplification by PCR.[18]

Inter-group comparison of both the groups showed that storing the teeth in a dry environment fetched a more correct interpretation of sex as compared to a wet environment. In a wet state, DNA in the pulp tissue may get degraded or fragmented and hence it is impossible to detect X and Y chromosome-specific DNA by PCR.[3]

In the present study, 100% diagnostic accuracy of gender determination by PCR was not achieved in both groups. The overall decrease in success rate on prolonged storage of teeth samples in both the groups could be due to loss of tissue during pulp extirpation, contamination of the pulp tissue while collection, time-lapse for the procedure, variation in the pulp volume, lack of adequate quantity and quality of DNA to perform PCR technique, and root resorption in the deciduous teeth.[19]

Other factors could be due to technical errors like loss or contamination of the samples during the storage period or lack of adequate care while doing the procedures that may lead to contamination of the biological sample which may result in wrong interpretation of the genetic makeup for the identification. Hence a strict quality assurance and quality testing program are mandatory. Hence in a forensic scenario, a short post-mortem interval and/or a dry environment would favour the preservation of DNA in the pulp, whereas a long PMI and/or wet environment would increase the reliance on hard tooth tissues for DNA.[20]


   Conclusion Top


It may be concluded that the pulp tissue obtained from deciduous teeth yielded good quality DNA sufficient enough to be amplified correctly by the PCR technique.

Both the method and period of storage of teeth play significant roles in gender determination. It may be concluded that teeth stored in an open environment showed a more correct interpretation of gender as compared to the samples stored in salt water.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Kholief M, El Shanawany S, Gomaa R. Sex determination from dental pulp DNA among Egyptians. Egypt J Forensic Sci 2017;7:29.  Back to cited text no. 9
    
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Willard HF, Waye JS. Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet 1987;3:273143.  Back to cited text no. 10
    
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Witt M, Erickson RP. A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction. Hum Genet 1989;82:271–4.  Back to cited text no. 11
    
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Duffy JB, Skinner MF, Waterfield JD. Rates of putrefaction of dental pulp in the northwest coast environment. J Forensic Sci 1992;36:1492-502.  Back to cited text no. 12
    
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Ionesiĭ AG. On the possibility of teeth gendering by cytological proceeding. Sud Med Ekspert 1980;23:27-8.  Back to cited text no. 13
    
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Lynch MJ, Raphael SS. Lynch's Medical Laboratory Technology. 3rd ed. Philadelphia: WB Saunders Company; 1976.  Back to cited text no. 14
    
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Vemuri S, Ramya R, Rajkumar K, Rajashree P. Influence of various environmental conditions on DNA isolation from dental pulp for gender determination using polymerase chain reaction. SRM J Res Dent Sci 2012;3:231-5.  Back to cited text no. 15
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Bender K, Farfán MJ, Schneider PM. Preparation of degraded human DNA under controlled conditions. Forensic Sci Int 2004;139:135-40.  Back to cited text no. 16
    
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Burger J, Hummel S, Hermann B, Henke W. DNA preservation: A microsatellite DNA study on ancient skeletal remains. Electrophoresis 1999;20:1722-8.  Back to cited text no. 17
    
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Mukherjee KK, Biswas R. Short tandem repeat (STRs) and sex specific Amelogenin analysis of blood samples from neurosurgical female transfused patients. J Clin Forensic Med 2005;12:10-3.  Back to cited text no. 18
    
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Haertig A, Krainic K, Vaillant JM, Derobert L. Medicolegal identification: Teeth and blood groups (author's transl). Rev Stomatol Chir Maxillofac 1980;81:361-3.  Back to cited text no. 19
    
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Correspondence Address:
M S Ravi
Head, Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, NITTE Deemed to be University, Mangalore - 575 018, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_507_21

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