folate is essential to the carbon transfer necessary for dna synthesis , cell division , and tissue growth .
the mthfr gene is located at chromosome 1p36.3 and is 2.2 kb in length with a total of 11 exons . within the mthfr gene a common c to t polymorphism
exists in exon 4 at position 677 , it is a point mutation that converts a cytosine ( c ) to a thymine ( t ) , resulting in an amino acid substitution of alanine to valine .
the t variant codes for a thermolabile enzyme leading to an activity of 65% in the heterozygous state ( ct ) and ~30% in the homozygous state ( tt ) , respectively .
several association studies have been conducted to assess whether an association exists between the polymorphism in the mthfr gene ( mim 607093 ) .
mthfr gene mutation has been related to many diseases including colon cancer , leukemia , vascular disease , depression , schizophrenia , migraine with aura , glaucoma , down syndrome , and neural tube defects .
skull development is a complex process that involves on going interaction between the bones of the skull and cranial soft tissues .
the cranial vault is comprised of intramembranous bones joined by the sutures of dense fibrous tissue that accommodate the growing brain .
bone is added at these sutures during growth , and the skull eventually ossifies completely .
well - known mutations in the fibroblast growth factor receptor-2 ( fgfr2 ) , twist1 , frem1 , lrit3 , efna4 and runx2 have not shown constant results with different ethnic population with nonsyndromic craniosynostosis . around ~20% of craniosynostosis cases
are syndromic , occurring with one or more additional major malformations caused by single - gene mutations in one of at least eight genes ( fgfr1 , fgfr2 , fgfr3 , twist1 , efnb1 , por , msx2 and rab23 ) , involving primarily the coronal sutures .
recently , rajagopalan et al . , have reported that common foliate gene variant , mthfr c677 t , is associated with brain structure in two independent cohorts of people with mild cognitive impairment .
they found that a very commonly carried variant in the mthfr gene , which is associated with high homocysteine levels in the blood , is significantly associated with brain structure variation , in particular with lower regional brain volumes , as per our knowledge , none of the studies had solely examined the association of mthfr gene c to t polymorphism in craniosynostosis patients in any part of the world . as a first step for a comprehensive genetic study on craniosynostosis
this family - based association approach has the advantage that it avoids possible ethnic stratification that may affect the conventional case - control design . to study the family - based association of the mthfr polymorphism in different categories of craniosynostosis patients .
to study the family - based association of the mthfr polymorphism in different categories of craniosynostosis patients .
to study the family - based association of the mthfr polymorphism in different categories of craniosynostosis patients .
this was a cross - sectional study in which 30 patients classified as apert syndrome , pfeiffr syndrome and nonsyndromic craniosynostosis patients with their family were recruited after obtaining clearance from the institutional ethics committee . in clinically suspected cases of craniosynostosis
an x - ray or a ct scan of the child 's skull was done .
a complete prenatal and birth history of the child , including family history of craniosynostosis or other craniofacial abnormalities was recorded .
three milliliter intravenous blood was collected from patients and from their family members ( father and mother ) in ethylenediaminetetraacetic acid - anticoagulated vacutainer for the purpose of the study after their written informed consent .
children with primary microcephaly ( secondary craniosynostosis ) and postural plagiocephaly and those with any chronic diseases or associated syndromic disorders , parents and relatives not giving consent were excluded from the study .
primers for mthfr gene were designed as described by reutter et al . and custom - synthesized primers ( sigma aldrich chemicals pvt . ,
bengaluru , india ) . polymerase chain reaction ( pcr ) for each sample was performed in 0.2 ml , thin - walled tubes using 20 g of dna , 2 - 5 pm of each primer , 200 mm dinucleotide triphosphates , 10 pcr buffer , 1.5 mm mgcl2 , and 0.5 units of dynazyme ii dna polymerase ( thermo scientific ) .
the pcr reaction was carried out in a t-100 dna engine ( bio - rad , hercules , ca , usa ) thermal cyclers under the following conditions initial - denaturation - 94c for 8 min , denaturation - 94c for 1 min , annealing - 63c for 1 min , extension - 72c for 1 min , final extension - 72c for 7 min 4c , forever repeated for 40 cycles .
the primer sequences were : modified primers ( f5-tcttcatccctcgccttgaac-3 ; r5-aggacggtgcggtgagagtg-3 ) according to frosst et al .
amplicons sized were verified by gel electrophoresis by running the pcr product on 2% agarose gel with the 100 bp maker [ figure 1 ] .
after successful amplification , a small aliquot ( 5 l ) of the mthfr reaction mixture was treated with 1 units of hinf i restriction enzyme ( neb ) . two percent agarose gel , polymerase chain reaction ( pcr ) product before hinf i restriction fragment length polymorphism ; lane 1 - 100 bp ladder ; lane 2 - blank ; lanes 3 , 4 , 5 , 6 , 7 and 8 - pcr product of 315 bp the amplified pcr products ( mthfr ) were subjected to hinf i restriction enzyme digestion at 37c for 1 h. the pcr products subjected to enzyme digestion was visualized on 3% agarose gel stained with ethidium bromide .
gel photography was done with bio - red gel doc system . for mthfr 677 ,
the pcr yielded a 315 bp product , which on digestion with hinf i produced a 176 bp and 139 bp fragments for tt condition ( homozygous polymorphic ) and a 315,176 and 139 bp fragments for ct condition ( heterozygous polymorphic ) .
an undigested product length of 315 bp was retained by the wild types [ figure 2 ] .
three percent agarose gel , hinf i restriction fragment length polymorphism analysis of methylenetetrahydrofolate reductase 677 ; lane 1 - 50 bp ladder ; lanes 2 , 3 , 4 , 6 and 8 - homozygous wild type ; lane 5 and 7 -heterozygous polymorphic
the amplified pcr products ( mthfr ) were subjected to hinf i restriction enzyme digestion at 37c for 1 h. the pcr products subjected to enzyme digestion was visualized on 3% agarose gel stained with ethidium bromide .
gel photography was done with bio - red gel doc system . for mthfr 677 ,
the pcr yielded a 315 bp product , which on digestion with hinf i produced a 176 bp and 139 bp fragments for tt condition ( homozygous polymorphic ) and a 315,176 and 139 bp fragments for ct condition ( heterozygous polymorphic ) .
an undigested product length of 315 bp was retained by the wild types [ figure 2 ] .
three percent agarose gel , hinf i restriction fragment length polymorphism analysis of methylenetetrahydrofolate reductase 677 ; lane 1 - 50 bp ladder ; lanes 2 , 3 , 4 , 6 and 8 - homozygous wild type ; lane 5 and 7 -heterozygous polymorphic
the amplified pcr products ( mthfr ) were subjected to hinf i restriction enzyme digestion at 37c for 1 h. the pcr products subjected to enzyme digestion was visualized on 3% agarose gel stained with ethidium bromide .
gel photography was done with bio - red gel doc system . for mthfr 677 ,
the pcr yielded a 315 bp product , which on digestion with hinf i produced a 176 bp and 139 bp fragments for tt condition ( homozygous polymorphic ) and a 315,176 and 139 bp fragments for ct condition ( heterozygous polymorphic ) .
an undigested product length of 315 bp was retained by the wild types [ figure 2 ] .
three percent agarose gel , hinf i restriction fragment length polymorphism analysis of methylenetetrahydrofolate reductase 677 ; lane 1 - 50 bp ladder ; lanes 2 , 3 , 4 , 6 and 8 - homozygous wild type ; lane 5 and 7 -heterozygous polymorphic
the allele and genotype frequencies of the mthfr of offspring and parents were as tabulated in table 1 .
these results suggest no significant association of markers with craniosynostosis in the cases . the distribution of mthfr 677 polymorphisms among mothers and babies . it was observed that for mthfr c677 t , three out of four cases were in the group pertaining to both mother and baby being carriers of polymorphic variant . in two cases , both fathers and mother carried a polymorphic variant to the patient suggesting that maternal mthfr c677 t polymorphism may be a genetic risk factor for craniosynostosis .
the folate metabolism pathway plays an important role in dna methylation , dna synthesis , cell division , and tissue growth , especially in the rapidly developing cells .
thus , a defective folate metabolism could result in an impaired dna synthesis or dna methylation involved in the craniosynostosis .
t + c. 677c > t in the methylene tetrahydrofolate reductase ( mthfr ) gene is associated with higher than normal levels of homocysteine , which may increase risk of thrombosis ( mthfr thermolabile variant thrombophilia ) .
in addition , this mutation / polymorphism is considered a mthfr thermolabile variant and is associated with hyperhomocysteinemia , increased cardiovascular risk , increased risk of neural tube defects , and increased risk of preeclampsia .
furthermore , mutations in the mthfr gene may cause mthfr deficiency / homocystinuria . to the best of our knowledge ,
the present study is the first family - based association study between the mthfr gene and craniosynostosis .
the study of single nucleotide polymorphism mthfr c677 t polymorphism in craniosynostosis has not been reported yet .
our results showed that , based on single - marker frequency analysis , c677 t polymorphism was not associated with craniosynostosis . in summary , mthfr is associated with higher plasma homocysteine , a well - known mediator of neuronal damage and brain atrophy .
our results do not support the hypothesis that the mthfr 677crt polymorphism plays any role in the development of craniosynostosis .
however our results do suggest that most mutations were transferred through the mother to their babies .
when taking the results of posterior power calculations into account , they even contradict a causative role of this polymorphism within the bounds of its estimated effect size level . in complex genetic traits , exogenous factors often do play a relevant etiologic role , and if these factors are not prevalent in the population under investigation , the role of interacting co - causative genetic factors might be missed .
our findings revealed that c677 t polymorphism of the mthfr gene was not directly involved in the pathogenesis of craniosynostosis in our indian population . from the viewpoint of the findings of animal , clinical and pharmacological studies ,
however , the studies performed until date have produced inconsistent results . to shed light on the potential etiological role of mthf genetic variants in the pathophysiological mechanism of craniosynostosis ,
c667 t polymorphism of the mthfr gene is unlikely to play a role in the pathogenesis of craniosynostosis though maternal mthfr c677 t polymorphism may be a genetic risk factor .
 background:677c to t allele in the 5 , 10-methylenetetrahydrofolate reductase ( mthfr ) gene has been implicated in the etiology of various syndromes and nonsyndromic diseases but till date no direct studies have been reported with craniosynostosis.objectives:the aim was to study the family - based association of mthfr polymorphism in different categories of craniosynostosis patients.materials and methods : this was a cross - sectional study in which 30 patients classified as apert syndrome , pfeiffr syndrome and nonsyndromic craniosynostosis patients with their family were recruited . 
 a sample of 3 ml intravenous blood was taken from patients and from their family members ( father and mother ) in ethylenediaminetetraacetic acid - anticoagulated vacutainer for the purpose of the study . 
 genomic dna was extracted from peripheral blood lymphocytes by phenol chloroform extraction method . 
 primers for mthfr gene were designed . 
 the polymerase chain reaction was carried out . 
 after successful amplification , a small aliquot ( 5 l ) of the mthfr reaction mixture was treated with 1 units of hinf i restriction enzyme ( neb ) . 
 results were obtained and compiled.results:a total of 30 patients / participants with craniosynostosis of indian descent and their parents formed the study group . 
 the genotyping did not confirm an association between the mthfr 677c to t polymorphism and between different categories of craniosynostosis . 
 when comparing the offspring of mothers statistically significant differences were found.conclusion:c667t polymorphism of the mthfr gene is unlikely to play a role in the pathogenesis of craniosynostosis though maternal mthfr c677 t polymorphism may be a genetic risk factor . 
Introduction
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Objectives
Materials and Methods
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Restriction fragment length polymorphism
Results
Discussion
Conclusion