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 Table of Contents  
Year : 2017  |  Volume : 4  |  Issue : 1  |  Page : 9-14

Linkage evidence and methylation at 2q region in nonsyndromic cleft lip and/or palate of Malay population

1 Reconstructive Science Unit, School of Medical Sciences, Health Campus, Universiti Sains , 16150 Kubang Kerian, Kelantan, Malaysia
2 Human Genome Centre, School of Medical Sciences, Health Campus, Universiti Sains , 16150 Kubang Kerian, Kelantan, Malaysia

Date of Web Publication2-May-2017

Correspondence Address:
Wan Azman Wan Sulaiman
Reconstructive Science Unit, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2348-2125.205413

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Introduction: Nonsyndromic cleft lip and/or palate (NSCLP) occurs as a result of multifactorial determinants, involving both genetic and environmental factors. Several candidate genes associated with NSCLP have been discovered through genetic approach, but there is paucity of studies focusing on epigenetic determinants in NSCLP. We are interested to reveal linkage evidence of SATB2 at 2q region in large-extended NSCLP families of Malay population and its methylation activity in causing cleft formation. Materials and Methods: Eight large-extended families were included in this study. Microarray analysis was carried out and genome-wide linkage was determined using GeneHunter Multipoint Linkage Analysis v2.1r5. SATB2 methylation was tested on 100 NSCLP patients by DNA sequencing. Results: Genome-wide linkage analysis has revealed significant nonparametric linkage score and suggestive logarithm of the odds (LOD) score at 2q region in family 50 and family 100. Genome-wide heterogeneity LOD score of 2.63 and α =0.122 were found in total families at 2q33.1-q35 region. Significant copy number loss (P < 0.05) in NSCLP family compared with the normal control supports the linkage evidence of SATB2 in those families with positive linkage. Epigenetic testing found SATB2 unmethylation at DNA promoter region. Discussion: Linkage evidence and significant low copy number of SATB2 in NSCLP family of Malay population confirmed that genetic factors play a major role in causing cleft defects. SATB2 unmethylation could not support the epigenetic occurrence in causing craniofacial deformities. Conclusions: Linkage evidence and significant low copy number of SATB2 in NSCLP family of Malay population confirmed that genetic factors play a major role in causing cleft defects. SATB2 unmethylation could not support the epigenetic occurrence in causing craniofacial deformities.

Keywords: Epigenetic, linkage, microarray, nonsyndromic cleft lip and/or palate

How to cite this article:
Shah NS, Sulong S, Wan Sulaiman WA, Halim AS. Linkage evidence and methylation at 2q region in nonsyndromic cleft lip and/or palate of Malay population. J Cleft Lip Palate Craniofac Anomal 2017;4:9-14

How to cite this URL:
Shah NS, Sulong S, Wan Sulaiman WA, Halim AS. Linkage evidence and methylation at 2q region in nonsyndromic cleft lip and/or palate of Malay population. J Cleft Lip Palate Craniofac Anomal [serial online] 2017 [cited 2021 Oct 16];4:9-14. Available from: https://www.jclpca.org/text.asp?2017/4/1/9/205413

  Introduction Top

Orofacial clefts (OFCs) are congenital malformations that comprise a large fraction of human congenital disabilities that affect the lip and/or palate.[1] Cleft lip and/or palate (CLP) are immediately recognizable disruptions of normal facial structure.[2] Several studies have reported that Asian and Native North American descent populations with prevalence of 2/1000 births have highest birth prevalence compared to the other populations.[3],[4] Several candidate genes have been reported to play a role in OFC including MSX1, SATB2, FGF, and IRF6.[5],[6],[7],[8] Main focus of this study is to discover evidence of linkage at chromosome 2q, particularly SATB2 in multiplex families of nonsyndromic CLP (NSCLP), and to investigate its methylation activity to be associated with craniofacial defects among the Malays.

Although SATB2 is known as one of the candidate genes to NSCLP, none of the genes seem to play a major role to this deformity due to population variation.[9] The first linkage study was done by Beiraghi et al. that reported the possible gene in 4q region tested on a single five-generation family of cleft lip/palate (CL/P) but later reported to have no significant evidence for linkage in 56 families tested.[10],[11] Linkage studies revealed several candidate regions that have been studied in different populations.[12],[13],[14]

As OFC occurs as a result of multifactorial determinants, epigenetic is one of the environmental effects that could also contribute to the cleft formation. In eukaryotes, epigenetic occurs by silencing the mechanism through reversible biochemical modifications of DNA and associated proteins without altering DNA sequence, whereas DNA methyltransferase catalyzes the reaction of cytosine to form 5-methylcytosine by an addition of a methyl group.[15],[16],[17] Long-term gene silencing occurred due to DNA methylation; hence, it correlates the DNA methylation to the inhibition of gene expression.[15] Since both genetic and environmental factors play a role in craniofacial deformities, we are interested to discover the functional role of SATB2 at 2q region from genetic and epigenetic findings among the Malays. Therefore, we could clarify SATB2 mutation presence as a result of either one or both factors.

  Materials and Methods Top


Eight large-extended families with a history of NSCLP were included while 100 NSCLP patients were included for epigenetic analysis. All the subjects were recruited from Hospital Universiti Sains Malaysia and blood withdrawal was undertaken. Healthy controls with no family history of OFC were included as a normal control. This study was approved by the Research Ethics Committee (Human) of the Universiti Sains Malaysia, and written informed consent was obtained from all the subjects.

Inclusion and exclusion criteria

All family members either affected with cleft lip, CL/P, or cleft palate (CP), or healthy individuals were included in this study. Patients were first screened by specialists of plastic surgery. Any major abnormalities or syndromes detected during the screening were excluded.[18] Patients with one minor anomaly such as low-set ears, hypertelorism, clinodactyly, and single palmar crease were included.

Genome-wide linkage analysis

Microarray analysis

Genomic DNA was isolated from blood samples using the QiaAmp DNA Blood Mini Kit (USA), following the manufacturer's instructions. Extracted DNA of the family members of NSCLP was genotyped using Illumina Infinium Human Linkage-24 Beadchip (Illumina, California, USA). Following DNA denaturation, the samples were hybridized, fragmented, and precipitated before the staining process.[18] Data were analyzed using Illumina's GenomeStudio Genotyping Module. Markers with a minor allele frequency >5% and call rate higher than 95% were used in the analyses.

Confirmation by copy numzber variation assay real-time polymerase chain reaction

Copy number variation (CNV) was carried out on the two selected families (family 50 and family 100) showed suggestive or significant linkage, healthy control as a normal and commercialized human control as a calibrator. Specific SATB2 primer-probe pair was designed; Homo sapiens SATB2 National Center for Biotechnology Information (NCBI) location: Chr.2:200134223-200335989; cytoband: 2q33.1d (Hs07541174_cn). CNV was performed using TaqMan ® Genotyping Master Mix for absolute quantitation of copy number using real-time polymerase chain reaction (qPCR). RNase P was used as an endogenous control and NTC (reaction mixture without DNA template) as a negative control. qPCR was run using ABI 7500 system with the following parameter; hold at 95°C for 10 min, followed by 95°C for 15 seconds and 60°C for 1 min for 40 cycles.

Epigenetic status of SATB2 by sequencing

Bisulfite-treated DNA and polymerase chain reaction

Bisulfite conversion of DNA was done using Methyledge Bisulfite Conversion System (Promega, USA), following the manufacturer's instructions. Specific primers were designed using MethPrimer program (www.urogene.org/methprimer/) to produce a set of primer pair to detect methylated and unmethylated DNA sequences for SATB2. PCR amplification was performed using GoTaq ® G2 Hot Start Green Master Mix (Promega, USA) kit with a total volume of 25 μl. PCR conditions for both methylated and unmethylated primers were as follows: initial denaturation at 95°C for 2 min, denaturation at 95°C for 15 seconds, annealing at 61°C for 1 min, extension at 72°C for 1 min for 40 cycles, and final extension 72°C for 5 min.

DNA sequencing analysis

Purified PCR products were sequenced using BigDye Terminator V3.1 Sequencing Standard Kit (Applied Biosystems, Foster City, USA) as recommended by the manufacturer. The cycle sequencing was carried out in a total volume of 20 μl, containing around 40–90 ng of purified PCR products, 2 μl BigDye reaction premix, 4 μl BigDye sequencing buffer, 1 μl primer (10 μM), and distilled water. The cycle sequencing was run using ABI GeneAmp 9700 PCR System with initial denaturation at 96°C for 1 min, denaturation at 96°C for 10 seconds, annealing at 50°C for 5 seconds for 25 cycles, and extension at 60°C for 4 min. DNA sequences were analyzed using the Bioedit Sequence Alignment Editor Software v5.0.9 and were blasted with reference sequence.[19]

Bioinformatics analysis

For linkage analysis, easyLinkage Plus v5.08 software was used to analyze a large-scale single nucleotide polymorphism data.[20] It would check for Mendelian/non-Mendelian genotyping errors and Hardy–Weinberg equilibrium. GeneHunter Multipoint Linkage Analysis v2.1r5 was carried out on both parametric and nonparametric analysis with logarithm of the odds (LOD) and nonparametric linkage (NPL) score. The thresholds for genome-wide linkage analysis were categorized based on the criteria proposed by Lander and Lander and Kruglyak (1995).[21] LOD score was indicated as if 1.9 ≤ LOD <3.3 is a suggestive linkage and LOD ≥3.3 is a significant linkage. The allele sharing NPL score was categorized into three stages; score in between 2.2 and 3.5 is a suggestive linkage, 3.6–5.3 is a significant linkage, and ≥5.4 is highly significant linkage. Data output were then imported to NCBI web-based followed by GeneDistiller database to sort the genes according to the information obtained from the linkage data.

Statistical analysis

Triplicates of two independent experiments were carried out, and data were expressed as mean ± standard deviation. Differences between the groups (NSCLP and normal) were evaluated using Mann–Whitney U-test, and P< 0.05 was considered statistically significant. These values were calculated using the SPSS Statistics (SPSS 22, SPSS, Inc., New York, USA).

  Results Top

Linkage evidence in nonsyndromic cleft lip and/or palate families

Suggestive linkage peaks have been attained in family 100 with parametric LOD score of 2.12 and 2.18 at chromosomal region 2q31.2-q33.3, respectively [Table 1]. Genome-wide heterogeneity LOD (HLOD) score of 2.63 and α = 0.122 was also found in total families at similar 2q33.1-q35 region [Table 1]. The score for HLOD ≥1 and α ≤ 0.71 indicate that the proportion of families linked and showed the presence of genetic heterogeneity in this population. Nonparametric analysis attained suggestive and significant NPL score (2.49 and 3.69) at 2q34-q36.3 and 2q31.1-q35 in family 50 and family 100, respectively [Table 1].
Table 1: Linkage intervals with a heterogeneity logarithm of the odds score .1 was detected in total families, logarithm of the odds score >2 at chromosomal region 2 detected in family 100 and nonparametric linkage score >2 in family 50 and family 100

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Copy number variation analysis

Copy number calculated (CNC) value for the calibrator was 2.06 and most normal controls (5 out of 6) had CNC value 2.49 ≥ CNC ≥1.50 [Figure 1]. From nine members of family 100, only one nonaffected member (100-4) showed CNC <1.50 (CNC = 1.29) while the other four nonaffected members including four affected members; 100-P1, 100-P2, 100-3, and 100-14 had 2.49 ≥ CNC ≥1.50. Similarly, all the family members (n = 8) in family 50 including the two affected individuals (50-9 and 50-12) had 2.49 ≥ CNC ≥1.50 [Figure 1].
Figure 1: Bars indicated copy number calculated of SATB2 in family 100 (purple bars), family 50 (green bars), healthy control (orange bars), and calibrator (blue bar). Error bars represent the standard deviation of uncertainty in each sample tested

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Scatter plot data showed a distinct distribution of CNC between the target NSCLP families and normal controls [Figure 2]. The straight line depicted the normal copy number fell at two. SATB2 copy number for family 50 and family 100 was found scattered <2 meanwhile SATB2 copy number in normal controls were dispersed to >2. By comparing the CNC of SATB2 between family 50 (1.90 ± 0.12, P = 0.039) and family 100 (1.87 ± 0.24, P = 0.045) to the normal control group (2.18 ± 0.30), a significant decrease of SATB2 copy number was found in both families in comparison to normal, indicating that SATB2 copy loss was present among the NSCLP families.
Figure 2: Scatter plot indicated copy number calculated value in family 50 and family 100 in comparison to the normal controls

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SATB2 methylation

With DNA sequencing, target sequence that was aligned together with the reference sequence revealed high repetition of SATB2 unmethylation for all the target and normal controls. C to T (C > T) conversion was frequently observed on the sequence alignment in both groups as indicated in blue circles [Figure 3]. This sequence confirmed that no SATB2 methylation occurred.
Figure 3: Methylation analysis on SATB2. Mapping of reference (upper) and target (lower) sequence in SATB2 for comparison. Unmethylation status was identified as repetitive C > T conversions detected, indicating in blue circles

Click here to view

  Discussion Top

Linkage evidence at 2q31.1-q35 region

A suggestive LOD score in family 100 and an HLOD score for total family (HLOD = 2.63, α = 0.124) was attained on chromosome 2q region. In addition, highest significant NPL score in family 100 and suggestive NPL score in family 50 were also attained in chromosomal region 2q31.1-q36.3. GeneDistiller database extracted several genes fell within this chromosomal region including ZNF533, FSIP2, HOXD4, SUMO1, MSTN, NRP2, ABCA12, and ERBB4. The most common one that plays a role in craniofacial development was SATB2. Although SATB2 has been widely reported to be associated with clefting, the results have not been consistent among populations. Therefore, it was our interest to study SATB2 since it has never been reported in Malay population linkage studies to date.

Several genome-wide linkage studies conducted using multiplex families from various ethnic origins showed evidence of linkage at chromosome 2q.[6],[9],[20] The genome scan meta-analysis from 13 populations of NSCLP conducted by Marazita et al. yielded highly significant result in 2q32-q35 region that contains the gene for DNA-binding protein SATB2.[21] In addition, linkage evidence and linkage heterogeneity have been attained in two regions of chromosome 2 and 2q region, respectively, in 10 multiplex families at three different regions.[6] Nevertheless, a previous study on case-parent trios in three distinct populations has found SATB2 as a candidate gene for NSCLP.[20] Therefore, positive linkage found in the 2q region in NSCLP families of Malay population has provided an evidence of gene susceptibility on the loci.

Copy number loss of SATB2

CNVs is able in altering gene expression or gene dosage and disturbing gene sequence that finally lead to genetic defects and phenotypic variation.[22],[23] As anticipated, absolute copy number loss of SATB2 was significantly detected among the affected families compared to the healthy controls. This finding plausibly explained the significant role of SATB2 in normal craniofacial development, whereas any loss of it may cause cleft defects. Previous finding has reported that SATB2 lied within the deleted region, with low copy number of SATB2 being detected in an NSCLP offspring rather than in unaffected parents and healthy controls.[24]

SATB2 copy number loss detected among the NSCLP patients could also be caused by a de novo change. Previous studies had detected a de novo mutation through array comparative genomic hybridization method. A patient with CP had submicroscopic copy number alterations whereas de novo 4.5 Mb deletion on loci 2q33.1 region has been detected.[24] However, here, we did not further investigate whether the copy number loss occurs due to a de novo process or inherited from an affected/unaffected parent. In addition, previous report on decreased SATB2 copy number caused increased of cell death at the developing jaw primordial including palate and hindered regional development and therefore caused the craniofacial defects.[25] The loss of SATB2 copy number in the family of affected cleft increased the risk and susceptibility of SATB2 to cause OFC.

SATB2 methylation

Epigenetic alteration works by triggered changes in gene expression or inherited changes of phenotype through DNA methylation without altering the DNA sequences.[26] Aberrant DNA methylation of susceptible genes could result in gene silencing and spontaneously affect the normal biochemical pathway and palatal developmental process.[26] It has been said that methylation often detected in a promoter region of genes.[26] Meanwhile, two separate studies on human methylome and murine secondary palate methylome found different findings. Methylated regions of interest were found in intragenic regions so-called gene bodies which include exons, introns and 3'- and 5'-untranslated regions rather than in a promoter region.[26],[27] Interestingly, methylated promoter regions were found associated with decreased gene expression, but intragenic methylation correlated with increased gene transcription.[27]

SATB2 is a homeobox transcription factor that is known to play a role in nonsyndromic CP. It was first reported whereas translocation in 2q32-q33 region disturbed the gene regulation.[28] Two similar findings have been reported with regard to SATB2 mutation in both syndromic (patient with CP, osteoporosis, and mental retardation) and nonsyndromic CP cases.[9],[29] Several studies have reported deletion or balanced translocations at 2q32-q34 region with commonly shared features such as developmental delay, cleft/high palate, defects in jaw formation, feeding difficulties, and abnormal dentition, which may relate to NSCLP as well.[24],[30],[31] Recently, there is no report regarding SATB2 methylation in association with NSCLP formation. SATB2 unmethylation in all the targets and normal controls found in our study confirm the epigenetic event did not occur and hence has no role in craniofacial development.

  Conclusion Top

Finding more samples from families with strong NSCLP history and collecting the samples from all the affected and unaffected members were the limitations of our study. The limitation was obvious whereas the linkage evidence of SATB2 at 2q region was only attained in two families, indicating that different family had different causative gene. Significant linkage and low copy number of SATB2 in NSCLP family compared to the normal indicated that genetic factor is the causal factor for SATB2 in NSCLP formation. This study revealed SATB2 methylation did not affect craniofacial development and could not support the role of epigenetic process in association with OFC.


We are grateful to all the patients, parents, and families for their cooperation. We thank the staff of Reconstructive Science Unit, Universiti Sains Malaysia. The study was supported by Research University grant 1001/PPSP/812083. We also thank the scholarship SLAB/SLAI under Ministry of Education Malaysia and USM for supporting the student.

Financial support and sponsorship

The study was supported by Research University Grant (1001/PPSP/812083).

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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