Keywords
anterior encephaloceles - neural tube defects - polymorphism
Introduction
Anterior encephalocele (AE) is a rare congenital anomaly of the central nervous system
characterized by protrusion of the intracranial content beyond the normal confinement
of the cranium. It is a visible defect at the root of the nose and hence also called
as “sincipital” ([Fig. 1]). Sporadic in the western world, AE cases are most frequently reported from the
developing nations of Southeast Asia where the incidence rate varies between 1 in
3,000 and 1 in 5,000,[1] but the possible explanation for its “remarkably specific geographical distribution”
is still unknown. Several studies have implicated genetic predispositions, nutrition,
and environmental factors as risk factors of neural tube defects (NTDs).[2]
Fig. 1 A child with frontoethmoidal encephalocele.
Two polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene—the
C677T (rs1801133 C > T) and A1298C (rs1801131 A > C)—have been frequently studied
in context of NTDs.[2]
[3]
[4]
[5]
[6] MTHFR regulates the folate one-carbon metabolic pathway. Both the C677T (Ala222Val)
and the A1298C (Glu429Ala) substitutions result in a decreased enzymatic activity.[7] In Assam there is a high incidence of AE, especially within its tea garden communities.[8] However, there are no data concerning the risk factors of AE for this community.
Therefore, this study was conducted to determine the association of MTHFR gene polymorphisms
with the risk of AE in this region.
Material and Methods
Study Population and Design
Forty-one AE patients, who were surgically operated and followed up in the Department
of Pediatric Surgery, consented to participate in this matched case-control study.
The cases were classified based on anatomical finding according to the system described
by Suwanwela and Suwanwela[9] into nasofrontal, nasoethmoidal, and naso-orbital encephalocele. We also recruited
82 age- and sex-matched apparently healthy kids who had visited the department.
To study the probable risk factors, we administered a precoded close-ended questionnaire.
We accessed the nutritional status of all the volunteers by measuring their body mass
index (BMI). Parental BMI was calculated by dividing weight in kilograms by the height
in meters squared (kg/m2) and classified according to the World Health Organization (WHO) BMI cutoff points.
In case of children, the BMI-for-age percentile was used to interpret “weight status
categories.” We used the Child and Teen BMI calculator (the calculator was available
at https://nccd.cdc.gov/dnpabmi/calculator.aspx). Of the 41 cases, only 21 cases approved for genetic analysis. One milliliter of
peripheral whole blood was collected from all volunteers and stored at −80°C until
analyzed.
MTHFR Polymorphism Analysis
Genomic DNA was extracted from whole blood samples using Wizard genomic DNA purification
kit (Promega). The MTHFR C677T and A1298C polymorphisms genotyping protocols were
adapted from Cicek et al.[7] A polymerase chain reaction (PCR) reaction mixture of 25 µL was set up using GoTaq
Hot start Green 2X master mix (Promega) containing nearly 200 ng template DNA. Primer
sequences for MTHFR C677T were 5′-GGT CAG AAG CT ATC AGT CT GAG-3′, and 5′-CTG GGA
AGA ACT CAG CGA ACT CAG-3′, which produced a 494 bp PCR product. The PCR cycles were
2 minutes at 94°C, followed by 30 cycles each of 30 seconds at 94°C, 30 seconds at
62°C, and 30 seconds at 72°C with final extension at 72°C for 7 minutes. The PCR product
was then digested using HinfI (Promega) at 37°C for 3 hours. When digested products were resolved on a 1.5% agarose
gel, wild-type alleles (677C, Ala) resulted in 100- and 394-bp fragments while the
variant alleles (677T, Val) resulted in 100-, 165-, and 229-bp products ([Fig. 2]). The MTHFR A1298C variant was amplified using primers 5′-AAG GAG GAG CTG CTG AAG
ATG-3′ and 5′- CTT TGC CT GTC CAC AGC ATG-3′ and generated a product sized 237 bp.
The cycling conditions were 5 minutes at 95°C, followed by 30 cycles each of 30 seconds
at 94°C, 30 seconds at 63°C, and 30 seconds at 72°C with final extension for 10 minutes
at 72°C. The PCR product was digested using MboII (Promega) at 37°C for an hour. A 15% acrylamide gel was used to resolve the digested
products in which wild-type alleles (1298A) resulted in 27-, 26-, and 183-bp fragments
whereas the variant alleles (1298C) resulted in 27- and 210-bp fragments ([Fig. 3]).
Fig. 2 PCR-RFLP analysis of MTHFR gene. The Ala allele is cleaved by HinfI to yield two
fragments (100 and 394 bp). The Val allele is cleaved by HinfI and yields three small
fragments (100, 165, and 229 bp). The heterozygote has four bands (394, 229, 165,
and 100 bp). PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.
Fig. 3 MTHFR A1298C PCR-RFLP results, 100 bp ladder, 15% PAGE. Yellow star indicates homozygous
mutant. PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.
Statistical Analysis
Date were analyzed using IBM SPSS Statistics 20 for Windows (IBM, SPSS Inc.). Initially
a univariate analysis was performed to identify the putative risk factors. These risk
factors were further analyzed using multiple logistic regression to study their independent
association with AE. p Value of ≤ 0.05 (two tailed) was considered for statistical significance.
Results
Among the 41 patients who participated in the study, 20 (48.8%) were male and 21 (51.2%)
were female. The mean age was 36 months, ranging from 1 to 14 years in both cases
and controls. [Table 1] lists the cases according to their anatomical locations. Nasofrontal AE was seen
mostly among the female cases whereas nasoethmoidal AE was predominant among males.
The MTHFR C677T genotype was in Hardy-Weinberg equilibrium (p > 0.8), but the MTHFR A1298C departed from the same (p = 0.0). Data from [Table 2] revealed the risk factors associated with AE. The univariate odds ratio (OR) for
MTHFR 677CT, MTHFR 1298AC, and 1298CC genotypes were 1.55 (95% confidence interval
[CI]: 0.49–3.51), 6.00 (95% CI: 1.65–21.88), and 2.57 (95% CI: 1.04–6.39), respectively.
The analysis also indicated that a low maternal BMI significantly increased the risk
of acquiring AE.
Table 1
Cases according to anatomical locations of encephaloceles
|
Sex
|
Total
|
|
Male
|
Female
|
|
Anatomical location of the hernia
|
Nasoethmoidal
|
Count
|
12
|
9
|
21
|
|
% within type of AE
|
57.1%
|
42.9%
|
100%
|
|
Nasofrontal
|
Count
|
6
|
10
|
16
|
|
% within type of AE
|
37.5%
|
62.5%
|
100%
|
|
Nasoorbital
|
Count
|
2
|
2
|
4
|
|
% within type of AE
|
50%
|
50%
|
100%
|
|
Total
|
Count
|
20
|
21
|
41
|
|
% within type of AE
|
48.8%
|
51.2%
|
100%
|
Abbreviation: AE, anterior encephalocele.
Table 2
Analysis of factors associated with AE
|
Category
|
Cases
n (%)
|
Control
n (%)
|
Crude OR
(95% CI)
|
p-Value
|
Adjusted OR
(95% CI)
|
p-Value
|
|
MTHFR genotypes,
n
(%)
[a]
|
|
677CC
|
14 (66.7)
|
31 (73.8)
|
Ref.
|
|
Ref.
|
|
|
677CT
|
7 (33.3)
|
10 (23.8)
|
1.55 (0.49–3.51)
|
0.29
|
2.07 (0.62–6.87)
|
0.24
|
|
677TT
|
0 (0.0)
|
1 (2.4)
|
0.00
|
1.0
|
0.00
|
1.0
|
|
1298AA
|
11 (52.4)
|
33 (78.6)
|
Ref.
|
|
Ref.
|
|
|
1298AC
|
4 (19.0)
|
2 (4.8)
|
6.00 (1.65–21.88)
|
0.01[b]
|
7.75 (1.15–52.24)
|
0.04[b]
|
|
1298CC
|
6 (28.6)
|
7 (16.7)
|
2.57 (1.04–6.39)
|
0.04[b]
|
1.54 (1.00–24.74)
|
0.76
|
|
MTHFR haplotypes
|
|
677C-1298A
|
22 (52.4)
|
58 (69.0)
|
Ref.
|
|
Ref.
|
|
|
677C-1298C
|
13 (31.0)
|
14 (16.7)
|
2.45 (1.0–6.02)
|
0.05[b]
|
0.94 (0.07–12.44)
|
0.96
|
|
677T-1298A
|
4 (9.5)
|
10 (11.9)
|
1.06 (0.30–3.71)
|
0.93
|
0.84 (0.15–4.78)
|
0.85
|
|
677T-1298C
|
3 (7.1)
|
2 (2.4)
|
3.96 (0.62–25.28)
|
0.15
|
1.37 (0.05–34.55)
|
0.85
|
|
BMI of mother
[c]
|
|
18.5–22.9
|
18 (45.0)
|
57 (71.2)
|
Ref.
|
|
Ref.
|
|
|
< 18.5
|
20 (50.0)
|
22 (27.5)
|
4.89 (4.13–11.21)
|
0.00[b]
|
6.30 (2.53–15.70)
|
0.00[b]
|
|
≥ 23
|
2 (5.0)
|
1 (1.3)
|
4.00 (0.52–30.61)
|
0.18
|
3.77 (0.42–34.12)
|
0.24
|
Abbreviations: AE, anterior encephalocele; BMI, body mass index; CI, confidence interval;
OR, odds ratio; Ref., reference.
a These risk factors were studied in 21 cases and 42 apparently healthy controls who
consented to participate in this study.
b Statistically significant.
c Insufficient data.
In the multivariate analysis, there was no association between the MTHFR C677T variant
genotypes and AE. The MTHFR 1298AC variant, however, was significantly associated
with the risk, 7.75 (95% CI: 1.15–52.24). The MTHFR C677T and A1298C haplotypes were
not associated with the risk of AE among all the study patients. An increase in OR
during the analysis indicated that low maternal BMI is an independent risk factor
of AE.
Discussion
In Assam, there is a high incidence of AE.[8] Till date only a few studies have been performed to detect the possible risk factors
of AE probably because this anomaly is predominant only in the socioeconomically backward
communities of Southeast Asia and Africa.[2]
[3]
[5]
[10]
[11] The present study is the first hospital-based case-control study to be performed
in India, which studies the association between MTHFR polymorphisms and BMI with the
risk of AE. It clearly indicates that children who carry the MTHFR 1298CC genotype
were at a greater risk of having AE. Underweight children were also at a significant
risk of developing AE.
Several studies have reported the C677T polymorphism as a significant risk factor
of NTD, but in case of AE, these results are conflicting.[3]
[12] In this study, we did not find any association between the MTHFR 677TT genotype
and AE, as the frequency of the 677T homozygous mutant genotype among cases was zero.
However, the 677CT genotype demonstrated a weak association. This observation is similar
to those of studies conducted in Indonesian and Javanese community where the frequency
of the 677TT genotype was close to zero.[12] Homozygosity for the 677T mutation among mothers increases the risk of recurrent
early pregnancy loss (REPL) by two- to threefolds in absence of folate supplementation.[13] The National Family Health Survey (NFHS-3) reports that the infant mortality rate
in Assam is very high and the consumption of iron folic acid among pregnant mothers
at only 13.7%.[14] The findings are suggestive of the significant impact of MTHFR polymorphisms in
the community.
As AE is a rare event, most of the previous associative studies use a heterogeneous
population of NTDs. In a recent review, Zhang et al suggested that MTHFR A1298C is
not an independent risk factor of NTD, but its effect could be influenced by low dietary
intake of folate and ethnicity.[3] This is the first study to report a significant association between the A1298C polymorphism
and the development of AE. In the multivariate analysis, both the genotypes, 1298AC
and 1298CC, demonstrated a positive association with the risk of AE, but only 1298AC
achieved statistical significance. Distinct ethnicity in this region and low folate
intake could explain this significant association. In this study, we used the BMI-for-age
percentile system to interpret “weight status categories” to find that underweight
children were at a significantly higher risk of having AE as compared with healthy
children. Underweight is an indicator of low socioeconomic power[15] which has been previously associated with AE.[11]
The present study hints toward a possible association between maternal BMI and the
risk of AE. In the univariate analysis ([Table 2]), low maternal BMI (< 18.5) was significantly associated with the risk of AE. Children
of obese mothers were also at an elevated risk, but as only a small fraction of mothers
in the cohort were obese, its association was not statistically significant. Although
these observations were consistent with those of Stothard et al,[16] according to Gao et al, maternal underweight, overweight, and obesity were not associated
with elevated risks of NTDs.[17]
In conclusion, this study suggests a strong association between MTHFR A1298C polymorphism
and the risk of AE in this community. The C677T polymorphism, however, did not constitute
a genetic risk factor in this study. Children who were underweight and born to mothers
having a low BMI were at an elevated risk of developing AE.
