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Gene Expression Profile Associated With Neural Tube Defects Among Infants Attending Muhimbili National Hospital And Muhimbili Orthopaedic Institute In Tanzania

Background Neural tube defects (NTDs) are severe congenital anomalies of the central nervous system. The specific cause is not known, though several factors including gene variants involving the folate metabolism have been implicated in the etiology. This study aimed at identifying the gene expression profile of selected genes known to be associated with NTDs among infants attending Muhimbili National Hospital (MNH) and Muhimbili Orthopaedic Institute (MOI). Methodology We conducted a matched case control study involving infants who were attending MNH and MOI during 6 months of the study period. Each case was matched with two controls by gestational age, sex and birth weight. Whole blood samples were collected from all study participants for genetic analysis. Total RNA was isolated using Qiagen RNA blood mini kit, and reversed transcribed into complementary DNA (cDNA) using Super Script II Reverse Transcriptase cDNA Synthesis kit. Real-time polymerase chain reaction was performed on extracted cDNA by a Light Thermal Cycler 480 machine using specific primers for studied genes to determine their expression levels. Results were analysed by GraphPad Prism 5Software, using Student t-test, and Bonferroni post hoc statistical tests. A p value of < 0.05 was considered to be statistically significant. Results The study recruited 50 cases and 100 controls. Among eight studied genes, we found significantly low expression of Methylenetetrahydrofolate reductase [MTHFR] gene among cases than controls (p=0.006). Expression of others genes were having variations and expressed at very low levels in both cases and controls. Conclusion genetic


Introduction
Neural tube defects (NTDs) are congenital malformations of the central nervous system including: the 3 brain, spinal cord, and skull. These result from failure of the neural tube to close properly during fetal embryogenesis around the third and fourth week of conception (1). Globally, 300,000 babies are born with NTDs per year (2),with the prevalence ranging from 0.2 to 10 per 1000 live births in specific geographical locations (3). In Tanzania, in the study done by Kishimba et al in 2012,NTDs were reported to have the highest incidence of 9 per 10,000 live births among selected external structural defects. (4).
The majority of NTDs have unknown etiology, involving complex interactions of genetic, nutritional and environmental factors (5). Studies have shown that genetic mutations involving the folate metabolism and planar cell polarity (PCP) signaling pathways may be associated with an increased risk of NTDs (6). Gene variants that reduce the 'efficiency' of folate one-carbon metabolism have also been shown to increase the risk of NTDs. The Methylenetetrahydrofolate reductase (MTHFR) polymorphism C677T (rs1801133) has also been found to be associated with a 1.8-fold increased risk of NTDs, although this predisposition was detectable only in non-Hispanic populations (7). Similarly, in a study done in the Irish population, Kirke et al also found that both the homozygous (TT) and heterozygous (CT) genotypes for MTHFR C677T are associated with an increased risk of NTDs (odds ratio of 1.52 and 2.56 respectively) (8).In contrast, Hayati et al found no association between MTHFR C677T gene and NTDs in the Malaysian population as the homozygous genotype was absent in both cases and control groups (9). Another significant risk factor detected is the Methylenetetrahydrofolate dehydrogenase (MTHFD-1) R653Q variant (rs2236225), a trifunctional enzyme that catalyses the conversion of tetrahydrofolate to 5,10-methylenetetrahydrofolate (10). Polymorphism in the Methylenetetrahydrofolate dehydrogenase-1-like(MTHFD-1-L), the gene for mitochondrial 10-formyl-tetrahydrofolate synthetase is also associated with an increased risk of NTDs (11).
Other genes such as Aminomethyltransferase (AMT) and Glycine dehydrogenase (GLDC) that encode enzymes of the glycine cleavage system have also been found to harbour a number of missense mutations resulting into NTDs. In the study done by Narisawa et al in 2011, two AMT gene variants c.589G>C(D197H) and c.850G>C(V284L) were identified in anencephaly and spinal bifida patients respectively, but they were absent in controls (12).  (15). Therefore, the evidence is highly suggestive of a genetic component as a contributory risk factor for NTDs.
There is limited data on genetic etiology of NTDs in sub Saharan Africa of which Tanzania is part.
Therefore, we conducted this study to determine gene expression profile for eight genes which have been implicated in NTDs causation from previous studies. The aim was to determine the genetic contribution to NTDs in our setting, which may serve as a basis for genetic counseling of couples, so as to reduce the incidence of NTDs.

Materials And Methods
A hospital-based matched case control study was conducted over a period of six months from November 2016 to April 2017 at the Muhimbili National Hospital and Muhimbili Orthopaedic Institute in, Dares Salaam Tanzania. All infants who were receiving neonatal, general paediatrics, and neurosurgical services, during the study period were eligible.
Sampling procedure and sample size: A minimum of 50 cases and 100 controls were enrolled. A case was defined as infant with any type of NTDs and controls were those without detectable birth defects who met matching criteria. Consecutive sampling was used to enroll the cases until the sample size was reached. Two controls who matched a case by gestational age, birth weight and sex and who met the inclusion criteria were also selected using consecutive sampling among infants attending MNH/ 5 MOI who had no obvious birth defects. Infants who met inclusion criteria but were very sick or whose mother did not consent were excluded.
Outcome variable: In this study, the dependent variable was the presence of the NTDs among the infants whilst for the independent variables; we selected eight genes which have been implicated from previous studies in NTDs etiology as shown in the Table 1.
Blood collection and laboratory procedures: We collected whole blood samples from all infants in the study for genetic analysis at MUHAS Genetic Laboratory. Blood samples were taken under aseptic technique using a 2milliliter syringe and 1milliliter volume of blood was collected into an EDTA vacutainer tube. The vacutainer were clearly labeled with the study identification number, time and date when the sample was drawn and sent to the laboratory in a cooler box within 2 hours of drawing the blood. In the laboratory, the samples were stored at -80ºc freezer to ensure preservation of genetic materials before analysis.

RNA extraction and RT-PCR analysis:
Total RNA for all 150 infants were prepared from the blood samples using QIAamp RNA Blood Mini Kit (Qiagen) following the manufacturer's recommendations, 1μg of the total RNA was reverse transcribed into cDNA using Super Script II Reverse Transcriptase First-Strand cDNA Synthesis kit in accordance to the manufacturer's protocol (Invitrogen). A real-time polymerase chain reaction(RT-PCR) was performed on extracted cDNA by a Light Cycler 480 Real Time PCR system (Roche) according to the manufacturer's protocol using primers indicated in the Table 1, and 2x SYBR Green Master Mix (Roche). GAPDH was used as the house-keeping gene and the results were analyzed using the delta-delta Ct method (2-ΔΔ Ct method) and presented as relative gene expressions (16).The PCR reaction was performed using standard RT-PCR conditions: 50°C for 2minutes, 95°C for10minutes, followed by 40 cycles of 95°C for 30seconds and 60°C for 1minute.
Data analysis:Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA) using the standard two-tailed Student's t-test for two data sets and ANOVA followed by Bonferroni post hoc tests for multiple data sets. A p-value of < 0.05 was considered to be statistically significant.

6
A total of 50 cases and 100 controls participated in this study. Most of the infants were <1month old (74%) with almost equal male to female ratio (52% male versus 48% female). The mean age of the participants was 1.78 ±3.2 months with an age range of 1 to 11 months. Majority of the infants (92%) were delivered at term, with 66% having normal birth weights as shown in Table 2.

Gene expression profile for eight (8) genes analyzed in the study population
We included eight genes of folate and planar cell polarity pathways which were selected from previously published NTDs researches as shown in the Table 1. After gene expression analysis, we found that there was a significantly low level of expression of the MTHFR gene amongst the NTDs cases than amongst the controls (p=0.006). On the other hand, there were a lot of variations in the expression levels of other genes and these were expressed at very low levels in both controls and cases as shown in Figure 1.

Discussion
This study was conducted to determine the gene expression profile associated with NTDs among infants attending MNH and MOI. Findings revealed a significant low expression of MTHFR gene among cases than controls.
NTDs result from failure of closure of the neural tube during fetal embryogenesis. Evidence from studies done in different population groups strongly suggest that MTHFR polymorphism (MTHFR C677T) is associated with an increased risk of NTDs (7,8). In this study, DNA sequencing was not done, which is required for detecting specific gene mutation. However, the significantly low expression of MTHFR gene in the cases group could suggest the possibility of having variants with low enzyme activity amongst the cases with NTDs. The MTHFR gene plays a key role in folate and methylation cycles which are required for essential processes including; DNA synthesis, DNA/protein methylation, cell division, and tissue growth; especially in the rapidly developing cells (5). Thus, a defective gene variant could result in an impaired DNA synthesis or DNA methylation involved in the neurulation process which is essential during neural tube closure.
The study found no association of NTDs and the expression levels of other genes such as VANGL1, VANGL2,Dvl2 and Dvl3.This is contrary to the studies done in China and Italy, which found a significant association between the incidence of NTDs and other genes in addition to MTHFR gene (10)(11)(12)(13)(14)(15). This could be partly explained by the fact that none of these studies were done in Africa and also the other studies included aborted fetuses. Therefore, there is the likelihood that, gene expressions can be influenced by genetic diversity among different population groups; as well as specific fetal developmental stages.
In this study, due to logistic reasons, DNA sequencing which is required for identifying specific genetic mutation/polymorphism could not be performed. Therefore we cannot link the gene expression profile with specific gene variant/mutation(s) which might be a risk factor among the NTDs cases.

Conclusion And Recommendation
In conclusion, our findings suggesting that a low expression level of MTHFR gene was a significant risk factor associated with NTDs amongst infants attending MNH and MOI. .
We recommend gene expression analysis to be done on mothers of infants with NTDs to identify those at risk; and further mutational analysis to be done to enhance genetic counseling and therefore to prevent recurrence of NTDs.   Figure 1 Relative gene expression profile for 8 selected genes analysed among 150 infants (NTD cases and controls) attending MNH and MOI