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 Table of Contents  
Year : 2021  |  Volume : 10  |  Issue : 4  |  Page : 199-204

Cytogenetic evaluation of orofacial clefts

1 Professor and Head, Department of Anatomy, MGM Medical College and Hospital, Navi Mumbai, Maharashtra, India
2 Professor and Head, Department of Oral and Maxillofacial Surgery, MGM Dental College, Navi Mumbai, Maharashtra, India

Date of Submission14-May-2021
Date of Decision03-Oct-2021
Date of Acceptance08-Oct-2021
Date of Web Publication28-Oct-2021

Correspondence Address:
Anjali Satyen Sabnis
Department of Anatomy, MGM Medical College and Hospital, Kamothe, Navi Mumbai - 410 209, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/NJCA.NJCA_60_21

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Background: Orofacial cleft (OFC) is one of the common congenital anomalies of the face which includes cleft lip and or cleft palate that causes abnormal appearance of the face. It leads to difficulty in speech and feeding, especially in new-borns. Genetic and nongenetic factors may lead to OFCs. An association of OFC to genetic cause was assessed in the study. Methodology: Conventional karyotyping was done in 133 patients of all the age groups with OFCs (syndromic and nonsyndromic) to find out chromosomal aberrations (CAs) in OFCs, Department of Oral and Maxillofacial Surgery, MGM Dental College, Navi Mumbai directed patients to cytogenetic laboratory. Project was started after taking consent and ascent from the patient and institutional ethical approval. Results: The incidence of CAs is 2.2%, and polymorphic variations are 3.7%. Out of 133 patients 47, XXY was seen in one case and trisomy 21 was seen in two cases, polymorphic variations like pericentric inversion in chromosome 9, 16qh + and 22 pstk + were observed in one case each and 9qh+ was seen in two cases. Conclusion: The incidence of genetic involvement in terms of CAs and polymorphic variations to OFC is low. The application of molecular technique in patients with OFC will help to find out genetic involvement. Genetic counseling and precise prenatal diagnosis will prevent the incidence of OFC.

Keywords: Cleft lip, cleft palate, karyotyping, orofacial cleft

How to cite this article:
Sabnis AS, Natrajan S. Cytogenetic evaluation of orofacial clefts. Natl J Clin Anat 2021;10:199-204

How to cite this URL:
Sabnis AS, Natrajan S. Cytogenetic evaluation of orofacial clefts. Natl J Clin Anat [serial online] 2021 [cited 2022 Jul 1];10:199-204. Available from: http://www.njca.info/text.asp?2021/10/4/199/329499

  Introduction Top

Orofacial clefts (OFCs) are highly prevalent abnormalities of the lip and or palate caused because of failure of partial or complete fusion of maxillary and mandibular process during the development of the face. Unilateral or bilateral failure of fusion of maxillary process with medial nasal process results into unilateral or bilateral isolated cleft lip (CL), respectively, and unilateral or bilateral failure of fusion of palatine process and primary palate leads to cleft palate (CP) with unilateral or bilateral CL, respectively. CP is caused because of nonjoining of two palatine processes. Oral clefts affect about 1–25/10,000 new-borns worldwide,[1] and the prevalence of isolated CP and CL with or without isolated CP is approximately 1/700 live births, with ethnic and geographic variation.[2] Depending on the location of the nonfusion, the extent of involvement of the lip and or palate varies giving birth to various classifications and severity scores.

Various factors such as smoking, alcohol, steroids, anticonvulsants, and less or no intake of folic acid and multivitamins in the first trimester of pregnancy are known to cause OFCs. Children with OFCs may be associated with involvement of other systems. The majority of these OFCs are nonsyndromic. Syndromic and nonsyndromic OFC may have shown connection with involvement of other systems in the remarkable number of cases.[3] Advances in syndrome delineation and medical genetics have demonstrated that most of the clinical entities are an expression of genetic variability. In single gene disorders, abnormalities are seen and group of abnormalities are associated with monogenic syndrome.[3] Four hundred and eighty-seven monogenic syndromes were diagnosed in the 2001 version of the London Dysmorphology database.[4] When the syndromes involve a clinically important change in structure and/or number of chromosomal, they are termed as chromosomal syndrome. Trisomy 13 and 18 and the 4p are chromosomal aberrations (CAs) which result into various syndromes which usually found with oral clefts.[5] Fogh-Anderson in 1942 documented first time the OFCs has a strong genetic component in the population.[6] Rationale based on genetics of nonsyndromic clefting is complex and not well realized. Gene-gene and gene-environment interactions probably play important roles.[7] Multiple studies have been documented the connection between chromosomal abnormalities in the form of number and/or structure and OFCs. Structural aberration such as duplication, deletion, and translocation are predisposing factors for OFCs. During structural aberration, there could be loss of genetic material or excess genetic material which hampers the normal process of face development. It is also observed that first degree relatives of affected individuals have 30%–40% risk of having CL and/or CP. The identification of CA in OFC will help to guide the patient, parent, and relative about the disease and for felicitous counseling of the parents.

This study was an attempt to find out the CA present in an individual with OFC. With proper genetic study, causative factor can be identified.

  Materials and Methods Top

This was an observational study conducted after obtaining Institutional ethical committee approval (letter no. 2018/4/19 dated on April 13, 2018). The present study consisted 133 patients with OFCs; in them 56 were male and 77 were female aged between 6 months and 25 years.

The inclusion criteria were as follows: Patients of all age groups and irrespective of gender with OFC, who have undergone surgical repair of the cleft or those who are yet to be operated, with or without systemic comorbidities, with or without other congenital abnormalities, prediagnosed syndromic participants from department of oral and maxillofacial surgery, MGM Dental College, Kamothe, Navi Mumbai, were referred to the Cytogenetic Laboratory of Department of Anatomy, MGM Medical College, Navi Mumbai.

Exclusion criteria: Participant who did not sign consent and or ascent form

Consent form was signed by patients and assent form was signed by parents in case of patient whose age is <8 years.

Plan of the study: This study was carried out by the Department of Anatomy, cytogenetics laboratory MGM Medical College and Department of Oral and Maxillofacial Surgery, at MGM Hospital, Kamothe and Navi Mumbai. Standard Pro forma was used to collect necessary information regarding each case which contains family history, maternal exposure to tobacco, alcohol, fertilizer, pesticides and consumption of drug during pregnancy period, folic acid supplementation, and consanguinity from all the patients. History of consumption of following drugs was taken, salicylates, sulphonamides, penicillin, tetracycline/chloramphenicol, sulfasalazine naprozen, anticonvulsants, glucocoticoid, phenytoin, antiepileptic drugs, valproic acid, thalidomide, and diazepam.

Process of karyotyping-Conventional Karyotyping was done by using 4 ml peripheral blood with the process of planting, harvesting, banding, and analysis.

  1. Planting

    • Label the tube with name of patient, date, and time of planting
    • Take 4 ml RPMI in culture tube
    • Add 1 ml FBS to it
    • Add 0.2 ml of blood to the tube
    • Add 20 units of PHA to the tube and incubate it at 37°C for 72 h.

  2. Harvesting

    • At 69th h, add 20 units Colchicine to the tube and incubate it for 45 min
    • Centrifuge the tube for 10 min at 1000 rpm after incubation
    • Discard the supernatant and add 6 ml of KCL to tube. Then incubate it for 20 min at 37°C
    • Later, add 3 drop fixative (1:3 acetic acid: Methanol) to the tube. Shake the sample well Centrifuge it for 10 min, discard the supernatant
    • Add 4 ml fixative and Centrifuge it for 10 min, discard the supernatant. Continue this steps till get a white pellet.

  3. Banding

    • After the white pellet is obtained, discard the supernatant and add fixative to the tube till the whole pellet is properly mixed
    • Using a dropper, drop 3–4 drops of the mixture on a slide from higher of 1 feet
    • Keep the slide on hot plate so that the excess evaporates and the slide is dry.

    Preparation of slide for G banding:

    • Slides will be removed from incubator and cooled to the room temperature
    • Trial banding will be done using 1–2 slides
    • Slides will be placed in trypsin solution for 5–15 s followed by Phosphate Saline Buffer (PBS) with serum for 2 min then will be washed with PBS
    • Slides will be stained with Giemsa stain for 8–10 min followed by washing with PBS
    • Slides will be dried and adequate banding will be confirmed
    • If appropriate banding is achieved, remaining slides will be stained
    • Observe the slide under the microscope.

  4. Analysis

    • Analysis was done by using IKAROS imaging software (Meta Systems, Germany) and Olympus microscope as per ISCN guidelines.

  Results Top

Out of 133 patients, 5 patients (3.7%) were with syndromic OFC in the present study [Table 1]. Out of 133 patients 77 females (57%) and 56 males (42%) showed OFC. Out of 133 patients, 3 patients with incidence of 2.2% showed numerical CAs and 5 patients with incidence of 3.7% showed of polymorphic variations i.e., inversion 9, 9qh+, 16qh + and 22pstk+ [Chart 1] and [Chart 2]. No significant association was found between mother's exposure to tobacco, Gutkha, consanguineous marriage, and OFC in the child.
Table 1: Incidence of syndromic and nonsyndromic orofacial clefts

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  Discussion Top

Genetic aberrations are known predisposing factors for syndromic and nonsyndromic OFC. Multiple studies on OFC and its relation with CAs proved that these congenital anomalies are either associated with structural and or numerical CAs or found accidently in the syndromes such as Down syndrome, Patau syndrome, Edward syndrome, and Klinfelter syndrome. Along with genetic causes, other factors such as maternal exposure to tobacco, alcohol, consumption of drugs in pregnancy period, low profile status, and consanguinity are found associated with OFCs.[8] In our study, these factors were looked upon through pro forma. It was found that out of 133 mothers, 23 gave a history of consumption of tobacco, gutkha and 4 mothers gave a history of exposure to urea, fertilizer during the pregnancy period. In four cases, third degree consanguinity was found associated with OFCs [Table 2]. None of the mothers gave a history of consumption of alcohol and any drugs during the pregnancy period. There was no family history of OFC was observed and all the mothers were supplemented with folic acid tablets. In the Chi-square tests that in exact 2-sided significance of Pearson Chi-square, the P is >0.05 (P = 0.206), whereas, in case of Fisher's exact test, P is >0.05 (P = 0.328) which clearly assumes that there is no significant association between the mother's exposure and orofacial defect in the child.
Table 2: Orofacial clefts and environmental factors

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In the present study, we found five cases out of 133 cases (3.7%) [Table 1] of CP with involvement of other systems [Table 3]. Karyotyping of all the cases was normal. Molecular karyotyping may be recommended to find out the cause in such cases. Children with OFC, mentally challenged and dysmorphism showed Emanuel syndrome or supernumerary der22t(11;22) duplication 4q27q35.2 with concomitant deletion 21q22.2q22.3 and duplication 12p13.33p13.32 with concomitant deletion 18q22.3q23.[9]
Table 3: Details of syndromic orofacial clefts

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Out of 128 cases of OFCs, 46 cases (35.9%) with CL, 38 cases (29.6%) with CP, and 44 cases (34.3%) with CL and palate were studied [Chart 3]. The prevalence of combined CLP was 40.9%, isolated CP was 35.5%, and of isolated CL was 23.5%.[10] It was noticed that the incidence of OFC was overall higher in females, particularly being the case with isolated CP while combined CL and CP was seen more commonly in males.[11],[12],[13]

Structural and or numerical CAs are associated with OFC. Change in the structure or a number of chromosome changes genetic dosage. Such changes when involve the process of the genes carrying out the development of lip and palate leads to malformation in the form of CL, CP, or both. Establishing a connection between the type and location of chromosomal abnormality and incidence of OFC can go a long way in defining the etiopathogenesis of this anomaly, suspect and identify syndromic involvement and most importantly serve as a basis to offer genetic counseling.

The incidence of change in the structure and number of chromosomes in patients with OFC is 3.6%[14] and in the current study was 2.3%. The incidence of structural and numerical aberrations in OFC ranges from 1.7% to 2.7% and 0.9% to 9%[14],[15],[16],[17],[18],[19],[20] [Table 4]. It was found in two cases of CLP trisomy 21 was found and in one case of CP. Klinfelter syndrome was found [Table 5] and [Chart 4]. It is noticed that numerical CAs are more than structural CAs in OFC. Any alteration in the number of chromosomes will lead to abnormal genetic material which might have diverted process of development. In the present case, there was an extra chromosome no 21 in two cases and extra X chromosome in one case [Figure 1]. Association of trisomy 21 and OFC is a frequent finding and is documented by many authors.[14],[17],[18] Association of XXY and CP is rare to occur, and in the current study, this association was observed. The first case was a 5-year-old male with nonsyndromic CL whose karyotype analysis revealed 47, XYY.[19] The presence of structural changes such as deletion, duplication, translocation, iso-chromosome, and ring chromosome may disfigure the functioning of gene leading to malformations. Such aberrations such as deletion[14] and translocation[18] are seen in OFCs. In the current study, we have not found any structural CA. Along with CA, polymorphic variations are seen in OFC. Polymorphic variations are enlargement in the length of heterochromatin of long arm of chromosome. These were considered as normal for long time.[21] Polymorphic variations, heterochromatin, and chromosomal polymorphism are considered as same. Heterochromatin is formed by tandemly organized highly repeated sequences of satellite DNA that do not encode proteins. Hence, chromosomal polymorphisms are considered normal Karyotypes.[22] Heterochromatin located in centromere has a significant role in spindle attachment and chromosome movement, meiotic pairing.[23] Alteration in chromatin may lead to centromere function with flaw and may create difficulty in pairing of homologous chromosomes causing abnormal events. Although there is dissension seen regarding polymorphic variations, their origin and result should not be ignored. Polymorphic variants occur in long arms of 1, 9, 16 and distal 2/3rd of long arm of Y chromosome oftenly.[21] Normal variants such as Yqh+, 16 qh+ were observed with the incidence of 7.2%,[14] whereas in the current study, it was 3.9%. In the current study, polymorphic variations like, 16qh+, 22pstk+ and inversion 9 were noted in one case while 9qh+ in 2 cases [Figure 2] and [chart 2]. Inversion 9 is found linked with CL and palate in 7 years girl,[24] whereas in the present case, it is found in 10 years boy with CL and pterygium.
Table 4: Chromosomal aberrations in orofacial cleft in different studies

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Table 5: Cytogenetic result of patients with orofacial clefts

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Figure 1: Shows chromosomal aberrations in OFC. (a) 47, XXY. (b) 47,XY + 21

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Figure 2: Shows polymorphic variation in OFC. (a) 46, XY, 22 pstk+, (b) 46, XY, 16qh+ (c) 46, XY, 9qh+, (d) 46, XY, inv (9)(p12q13)

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  Conclusion Top

Cytogenetic studies are helpful to find out the involvement of CA in OFCs. This provides some conclusive information for offering genetic counseling to the patients at risk.

Molecular study such as molecular karyotyping will be useful for those patients whose karyotyping is normal to find out genetic basis of OFCs. Working up on environmental factors such as parental exposure to drugs, chemicals, and deficiency of vitamins along with molecular technique application will pave the way for recognition of causative factors.


The authors would like to thank Dr. Gaurav Deshpande, Department of Oral and Maxillofacial Surgery, MGM Dental College, Kamothe, Navi Mumbai-410209 for referring patients with OFC to cytogenetic laboratory.

Financial support and sponsorship

This study was financially supported by MGM Medical College funded the project in the form of supplying chemicals and reagents for carrying out chromosomal analysis.

Conflicts of interest

There are no conflicts of interest.

  References Top

Mossey PA, Little J, Munger RG, Dixon MJ, Shaw WC. Cleft lip and palate. Lancet 2009;374:1773-85.  Back to cited text no. 1
Leslie EJ, Carlson JC, Shaffer JR, Feingold E, Wehby G, Laurie CA, et al. A multi-ethnic genome-wide association study identifies novel loci for non-syndromic cleft lip with or without cleft palate on 2p24.2, 17q23 and 19q13. Hum Mol Genet 2016;25:2862-72.  Back to cited text no. 2
Venkatesh R. Syndromes and anomalies associated with cleft. Indian J Plast Surg 2009;42 (Suppl):S51-5.  Back to cited text no. 3
Winter RM, Baraister M. London Dysmorpholgy Database, London Neurogenetics Database and Dysmorphology Photo Library on CD-ROM. Ver. 3. Oxford: Oxford University Press; 2001.  Back to cited text no. 4
Bergé SJ, Plath H, Van de Vondel PT, Appel T, Niederhagen B, Von Lindern JJ, et al. Fetal cleft lip and palate: Sonographic diagnosis, chromosomal abnormalities, associated anomalies and postnatal outcome in 70 fetuses. Ultrasound Obstet Gynecol 2001;18:422-31.  Back to cited text no. 5
Fogh-Andersen P. Inheritance of Harelip and Cleft Palate; Contribution to the Elucidation of the Etiology of the Congenital Clefts of the Face. Copenhagen: Munksgaard; 1942.  Back to cited text no. 6
Lees M. Familial risks of oral clefts. BMJ 2008;336:399.  Back to cited text no. 7
Little J, Cardy A, Munger RG. Tobacco smoking and oral clefts: A meta-analysis. Bull World Health Organ 2004;82:213-8.  Back to cited text no. 8
Shenoy RD, Shenoy V, Shetty V. Chromosomal abnormalities in syndromic orofacial clefts: Report of three children. Case Rep Genet 2018;2018:1928918. doi: 10.1155/2018/1928918.  Back to cited text no. 9
Pereira AV, Fradinho N, Carmo S, de Sousa JM, Rasteiro D, Duarte R, et al. Associated malformations in children with orofacial clefts in portugal: A 31-year study. Plast Reconstr Surg Glob Open 2018;6:e1635.  Back to cited text no. 10
Wyszynski DF, Sárközi A, Czeizel AE. Oral clefts with associated anomalies: Methodological issues. Cleft Palate Craniofac J 2006;43:1-6.  Back to cited text no. 11
Stoll C, Alembik Y, Dott B. Epidemiological and genetic study in 207 cases of oral clefts in Alsace, north-East France. J Med Genet 1991;28:325-9.  Back to cited text no. 12
Milan M, Astolfi G, Volpato S, Garani GP, Clementi M, Tenconi R, et al. 766 cases of oral cleft in Italy. Data from Emilia Romagna (IMER) and northeast Italy (NEI) registers. Eur J Epidemiol 1994;10:317-24.  Back to cited text no. 13
Jun-ichi AZ, Kohama Gi, Sasaki M. Cytogenetic studies in patients with cleft lip and/or cleft palate (IV)., lap. 1-1. Hum Genet 1978;23:161-6.  Back to cited text no. 14
Subrt I, Cervenka J, Krecek M. Cytogenetic study of cleft lip and palate. Cleft Palate J 1966;3:362-7.  Back to cited text no. 15
Abdulameer SJ, Hanash AA. Chromosomal analysis of types cleft lip and palate in the city of Kut. Eurasia J Biosci 2020;14:2007-10.  Back to cited text no. 16
Patel BM, Kodiyatar BB, Patel S. The karyotype analysis in cases of cleft lip and cleft palate. Scholars Int J Anat Physiol 2019;2:124-7.  Back to cited text no. 17
Hirakawa M, Adachi K. Cytogenetic studies in 100 patients with cleft lip and cleft palate. Kyushu Dent Soc 1970;24:73-85.  Back to cited text no. 18
Al Adsani AM. Cytogenetic studies on cleft lip and/or cleft palate in Kuwait. Egypt J Med Hum Genet 2003;4:35-44.  Back to cited text no. 19
Coco R, Penchaszadeh VB, Opitz JM. Cytogenetic findings in 200 children with mental retardation and multiple congenital anomalies of unknown cause. Am J Med Genet 1982;12:155-73.  Back to cited text no. 20
Hong Y, Zhou YW, Tao J, Wang SX, Zhao XM. Do polymorphic variants of chromosomes affect the outcome of in vitro fertilization and embryo transfer treatment? Hum Reprod 2011;26:6.  Back to cited text no. 21
Bhasin MK. Human population cytogenetics: A review. Int J Hum Genet 2005;5:83-152.  Back to cited text no. 22
Karpen G, Endow S. Meiosis: Chromosome behaviour and spindle dynamics. In: Endow S, Glover D, editors. Frontiers in Biology. Oxford: Oxford University Press; 1998.  Back to cited text no. 23
Rao BV, Kerketta L, Korgaonkar S. Pericentric inversion of chromosome 9[inv (9)(p12q13)]: Its association with genetic diseases. Indian J Hum Genet 2006;12:129-32.  Back to cited text no. 24
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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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