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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 3  |  Issue : 3  |  Page : 137-142

Non syndromic congenital malformations - a DNA study using comet assay


1 Assistant Professor of Anatomy, Mahatma Gandhi Medical College & Research Institute, Puducherry, India
2 Assistant Professor of Anatomy, P K Das Institute of Medical Sciences, Vaniamkulam, Kerela, India

Date of Web Publication21-Jan-2020

Correspondence Address:
Rijied Thompson Swer
Assistant Professor of Anatomy, Mahatma Gandhi Medical College & Research Institute, Pillaiyarkuppam,(Cuddalore-Pondy Main Road), Puducherry-607 402
India
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Source of Support: None, Conflict of Interest: None


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  Abstract 


Background and Objectives: The role of genomic instability resulting from chromosomal aberrations, gene mutations due to deletions, translocations and single gene defects is a known phenomenon leading to DNA damage. A deficient repair process is also attributed to the perpetuation of this damage. Placental insufficiency in pregnancy during late embryonic or early fetal period resulting in DNA damage gives rise to malformed phenotypes. An attempt was made to study the extent of DNA damage in non syndromic congenital malformations. Materials and Methods: A total of 20 children were studied. 10 of them, between 10 days to 5 years of age, presenting with non syndromic congenital anomalies formed the cases. An equal number of children matched for age criteria formed the controls. Lymphocytes collected from peripheral blood of these children were subjected to the standard comet assay, an highly sensitive, reliable, relatively inexpensive and reproducible single cell layer electrophoretic technique, where damaged DNA migrates out of the cell towards the anode forming a comet. The length of the tail is a measure of the DNA damage. Results: The malformations observed were those of urogenital, craniofacial and nervous systems. The mean comet tail lengths were 24.744 20.649 |im and 27.402 |im respectively. Comparing this to the mean tail length in controls with 1.992 fim, there was high statistical significance (P value <0.0001). Conclusion: Gene mutations, particularly involving Sex Region Determining (SRD) genes and Superoxide Dismutase (SOD) enzyme imbalances, have been implicated in these congenital malformations. Thus the comets seen in this study reflect the DNA damage due to the gene defects.

Keywords: comet scoring, migrating DNA, DNA damage, comet tail, gene mutation


How to cite this article:
Swer RT, Datta D, D'Silva MH. Non syndromic congenital malformations - a DNA study using comet assay. Natl J Clin Anat 2014;3:137-42

How to cite this URL:
Swer RT, Datta D, D'Silva MH. Non syndromic congenital malformations - a DNA study using comet assay. Natl J Clin Anat [serial online] 2014 [cited 2022 Aug 11];3:137-42. Available from: http://www.njca.info/text.asp?2014/3/3/137/297373




  Introduction Top


An association of chromosomal aberrations, gene mutations and involvement of multiple loci with variable combination of genes like Non Allelic Homologous Recombination (NAHR), Low Copy Repeats (LCR) and Complex Chromosome Rearrangements (CCR) have been implicated in the aetiology of congenital anomalies[1],[2]. Congenital anomalies cause approximately 21% of infant mortality of which 40% to 60% are of unknown aetiology and may be attributed to genetic causes[3]. These defects are classified as major and minor with the risk of major anomalies increasing proportionally with the presence of minor anomalies; thus minor anomalies often serve as a pointer to underlying major anomaly[3].

Congenital anomalies are labelled according to sequences, with four such patterns described. The first sequence is that of malformations, resulting from poor formation of tissues, presenting either as a single localised defect or at multiple sites. The second sequence results from abnormal forces acting on already formed tissues giving rise to deformities. The third sequence is that of disruptions due to destruction resulting from morphological alteration of tissues. The fourth sequence gives rise to dysplasia due to abnormal organisation of tissues[3].

Major malformations, accounting for 4% to 6% of these anomalies, result in a structural physical deformity of cosmetic and functional significance and half of these are observed at child birth and the other half by 5 years of age[4]. Minor anomalies, neither a life threat nor requiring medical or surgical intervention for survival, account for approximately 10% of newborns[5].

Some of the common anomalies occurring with varying grades of severity include cleft lip, cleft palate, club foot, congenital pyloric stenosis, congenital dysplasia of hip, ambiguous genitalia and cardiac defects[6].

The entire process of morphogenesis is programmed in a timely and sequential manner when the zygote undergoes cell divisions and differentiation begins to occur by activation or inactivation of certain genes, permitting cells to assume diverse roles[7],[8].

DNA damage is a common factor in these anomalies and there is limited literature on assessment of this damage in these diseases. The single cell gel electrophoresis or the comet assay is a reliable, highly sensitive, relatively inexpensive and reproducible technique in the quantification of the DNA damage where damaged DNA moves out of the cell[9]. Hence we attempt to measure the DNA damage in non syndromic congenital malformations using this method.


  Materials and Methods Top


With permission obtained from the Institute ethics committee for the study, 20 children presenting to the Out Patients Department of Paediatrics, JIPMER, Puducherry, from 2007 to 2009, were included in this study according to the following criteria.

Inclusion criteria

  1. Children with non syndromic congenital malformations less than 5 years of age formed our cases. A total of 10 such randomly selected cases were chosen.
  2. 10 children who had come for immunisation and were otherwise normal were matched for age with the above group and formed our controls.


Children with syndromic malformations were not included.

The peripheral blood of these children was subjected to the electrophoresis in the Cytogenetics Laboratory of Anatomy department, JIPMER. The protocol of Singh NP et al for conduct of comet assay was followed[9]. The following steps were performed under dim light to minimise artificial DNA damage.

  1. Collection of Peripheral venous blood- 2ml sample was taken from each child.
  2. Isolation of lymphocytes by centrifugation.
  3. Preparation of reagents
  4. Layering of slides
  5. Treatment with ly sing buffer
  6. Treatment with alkali
  7. Electrophoresis with low current
  8. Neutralisation
  9. Staining with Silver Nitrate
  10. Comet scoring.


Lymphocytes represent the general genomic instability by virtue of being rapid dividers. Treatment with alkali makes the technique more sensitive for detection of very low levels of DNA damage particularly those seen in single strand breaks[10]. Alow current being used prevents migration of normal DNA[9]. Staining with silver nitrate confers a degree of permanency of the stain allowing storage and scoring of comet metrics at a later date[11].

Comet scoring was done using a bright field transmission light binocular research microscope with photo micrographic attachment. The extent of DNA damage is related to the amount and length of DNA migration out of the cell forming the tail of the so called comet, the head representing the undamaged DNA. Thus comet tail length was calculated by subtracting the head diameter from the total length of the comet as shown in [Figure 1]. Fifty random cells per slide were scored and 1000 comets were studied; 500 belonging to cases and 500 to controls.
Figure 1: Showing calculation of comet tail length

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  Observations and Results Top


The cases were in the age group from 10 days to 4 years and the number of cases in each age group is shown in [Table 1]. The congenital anomalies encountered were mainly those of ambiguous genitalia followed by craniofacial defects and neural tube defects. The malformations according to age and gender are shown in [Table 2].
Table 1: No. of cases in each age group

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Table 2: Malformations according to age and gender

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The controls were 10 healthy children, matched for age with the cases and randomly chosen from the children presenting to the Paediatrics OPD for routine immunization and without any congenital anomalies.

Peripheral blood collected from these 20 children and subjected to comet assay as described above showed the formation of tails in the cases group. The mean comet tail length among the cases is shown in [Table 3]. Only the cases of ambiguous genitalia were subjected to karyotyping. Of them, only two children of 10 days and 4 years age showed the male karyotype. Hormonal profile done in them showed high levels of testosterone. The remaining four children showed the female karyotype. All the six cases of ambiguous genitalia had persisting labioscrotal swelling. The swellings were noticed to be more like bifid scrotum with the phallus buried. But the phalli were not equal in size, thereby also suggesting the possibility of a clitorimegaly. Only one swelling palpated revealed the presence of a soft structure inside it.
Table 3: Mean comet tail length in individual cases

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[Figure 2] shows a child with cleft Up and palate, [Figure 3] shows a child with meningocoele and [Figure 4] shows a child with ambiguous genitalia. Among the cases, as seen in [Table 3], the mean tail lengths of the children with ambiguous genitalia were significantly longer, followed by the child with neural tube defect (P value <0.0001). When the tail lengths of the cases were compared to those of controls, those of the cases were longer than the controls with statistical significance (P value <0.0001) as shown in [Table 4].
Figure 2: Child with cleft lip and cleft palate

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Figure 3: Child with meningocoele

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Figure 4: Child with ambiguous genitalia

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Table 4: Comparison of mean tail lengths between cases and controls

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The controls were 10 healthy children, matched for age with the cases and randomly chosen from the children presenting to the Paediatrics OPD for routine immunization and without any congenital anomalies.


  Discussion Top


In our study the cases of ambiguous genitalia, cleft lip, cleft palate and meningocoele seen are regarded as major malformations according to the classification assigned to them[4].

In the two children with ambiguous genitalia with male karyotype, and high levels of testosterone, the defect could be in the conversion of testosterone to dihydrotestosterone, the hormone that is responsible for formation of normal male external genitalia. 5 alpha reductase is the enzyme involved in the above conversion. The involvement of mutation in SRD5A2 (Sex Region Determination) gene of the Y chromosome is reported to cause sub functioning to non functioning of the enzyme; other loci reported are E57Q, G85D, G115D, S210F, G183S and P212X leading to masculization defects[12].

Neural crest cells are vital for the development of craniofacial region, great vessels, septae and valves of the heart. Defects of these cells give rise to a spectrum of diseases; Teacher Collins syndrome (mandibulo facial dysostosis), Robin sequence that includes Di George’s anomaly, velocardiofacial syndrome and conotruncal anomalies being ascribed to defective neural crest cell migration. Deficiency of SOD (superoxide dismutase) leading to accumulation of toxic free radicals in the neural crest cells induces cell damage by creating oxidative stress on them[3]. In our study, the three children with craniofacial anomalies were not part of any syndrome because they did not have any cardiac defects. Nevertheless, SOD enzyme imbalance has been reported in isolated craniofacial deformities[3]. The same holds true for causation of neural tube defects[3].

The DNA damage seen in the form of a comet is commonly ascribed to either single strand or double strand breaks. Formation of comets was seen in the cases group with the children of ambiguous genitalia having the longest tails, thus suggesting DNA damage. [Figure 5] shows the slide of a case with comets. There was no formation of comets in the control group suggesting no DNA damage. [Figure 6] shows the slide of a control. The comet assay was done by us using high alkaline buffer. Hence the damaged DNA in the migrated form could be from an alkali labile site of the DNA strands, probably from of a single strand break. Base excision repair, nucleotide excision repair and mismatched repair take place as an alternative mechanism for the repair process in the case of single strand breaks; this mismatched repair results in damaged DNA[5]. Resynthesis of DNA polymerase and DNA ligases are necessary for the final nick sealing stage in the removal of damaged DNA and defects related to the above would lead to accumulation of such damaged DNA. Single base damage is generally known to be caused by oxidation, alkylation, hydrolysis and deamination[5]. Probably it could be placental insufficiency during the late embryonic or early fetal period as the triggering factor for DNA damage resulting in malformations.
Figure 5: Slide of case showing comets

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Figure 6: Slide of control with no migrating DNA

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  Summary and Conclusions Top


In our study significant changes were seen in the formation of comets in children with non syndromic congenital malformations.

  1. The mean comet tail length of the cases was significantly longer than those of the controls.
  2. The mean comet tail length of the children with ambiguous genitalia was the longest, followed by that of the child with meningocoele and the children with craniofacial anomalies.


Thus it is seen that DNA damage due to gene mutations has been a perpetuator of congenital malformations in tune with reported literature. A correlation with karyotyping, hormonal profiling, enzyme assays and imaging modalities widens the scope of the study and helps in defining various anomalies.

Acknowledgment

We express our sincere thanks to Dr. Parkash Chand, Prof. Department of Anatomy, Dr. Vishnu Bhat, Prof. Department of Pediatrics and Dr. Ramachandra Rao Head of the department of Anatomy, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry for their timely advice and guidance to undertake this study.



 
  References Top

1.
Lupski JR. Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet 1998; 14:417-422.  Back to cited text no. 1
    
2.
Vissers LELM, Stankiewicz P, Yatsenko SA, Crawford E, Creswick H. Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies .Hum Genet, 2007;121:697-709.  Back to cited text no. 2
    
3.
Sadler TW (Ed). Langman’s Medical Embryology, 10th ed. Lippincott Williams and Wilkins. Published by Wolters Kluwer Health (India) Pvt. Ltd, New Delhi,2006: 111.  Back to cited text no. 3
    
4.
Kumar V, Abbas AK, Fausto N (Eds). Robbins and Cotran’s Pathologic Basis of Disease. 7th edition. Elsevier Saunders. Philadelphia, 2005:470.  Back to cited text no. 4
    
5.
Turnpenny P, Ellard S. Emery’s elements of medical genetics. 13th ed. Churchill Livingstone Elsevier. Edinburg, 2007:237.  Back to cited text no. 5
    
6.
Aase JM. Diagnostic dysmorphology. University of New Mexico School of Medicine. Albuquerque. New Mexico, 1990.  Back to cited text no. 6
    
7.
Kenneth LJ. Smith’s recognizable patterns of human malformation.Sixth Edition. Elsevier Saunders. W.B. Company. Philadelphia, 2006:783.  Back to cited text no. 7
    
8.
Epstein CJ, Erickson RP, Wynshaw-Boris A (Eds). Inborn errors of development. The molecular basis of clinical disorders of morphogenesis. Oxford University Press. New York, 2004:3-9.  Back to cited text no. 8
    
9.
Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988; 175:184-191.  Back to cited text no. 9
    
10.
Singh NP, Stephens RE, Schneider EL. Modifications of alkaline microgel electrophoresis for sensitive detection of DNA damage. Int J Radiat Biol. 1994; 66(1): 23-8.  Back to cited text no. 10
    
11.
Nadin SB, Vargas-Riog LM, and Ciocca DR.A silver staining method for Single-cell Gel Assay. J HistochemCytochem. 2001;49:1183-1186.  Back to cited text no. 11
    
12.
Des Groseilliers M, Beaulieu Bergeron M, Brochu P, Lemyre E, Lemieux N. Phenotypic variability in isodicentric Y patients. Clin Genet. 2006; 70(2): 145-50.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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