Abstract
The purpose of this communication is to report an unusual case of an infant affected with both neural tube defect (NTD) and Down syndrome (DS). Genetic risk factors in the mother were investigated. As impaired folate metabolism is implicated in the etiology of NTD and DS, folate gene polymorphisms were determined in the case-mother. These included methylene tetrahydrofolate reductase gene (MTHFR) 677C>T, methionine synthase gene (MTR) 2756A>G, betaine homocysteine methyl transferase gene (BHMT) 742G>A, thymidylate synthase gene enhancer region (TSER) – 3 or 2 tandem repeats of 28-bp and TS gene, 6-bp deletion in 3’ untranslated region. The genotype distribution and minor allele frequencies were also determined in a group of female control subjects. The genotypes of case-mother was MTHFR 677CT, MTR 2756AG, TSER 3R2R, TS D0D6 and BHMT 742AA. The BHMT 742AA genotype, in combination of genotypes of case-mother at five, four or three polymorphic loci, was either absent or present in a small number of control women. The case mother was a life-long vegetarian. Although the precise maternal risk factor(s) cannot be pin-pointed, it is possible that BHMT 742AA genotype coupled with restricted intake of those folate micronutrients present chiefly in foods of animal origin may play a role.
Introduction
The worldwide prevalence of neural tube defects (NTD) is estimated to be 300,000 to 400,000 live births annually with geographical variations such as about 1 in 1000 in the United States [1,2] and averagely about 3.63 per 1000 in India [3,4]. The prevalence of Down syndrome (DS) is estimated to be about 1:800 1:1000 live births annually [5, 6]. The major affliction of DS-affected children is the presence of moderate to severe mental retardation. Neural tube defects are a range of congenital malformations associated with the failure of the neural tube to close properly during early embryonic development leading to severe physical disability or death of the newborn. Clinical research evidence suggests that abnormal folate metabolism with impaired biological methylation that regulates gene expression may constitute maternal risk factors for NTD or DS [7, 8]. Due to this possible common etiology involved in the two birth disorders, investigations were carried out to explore any familial link between NTD and DS. A study in two series of families from separate countries, one with a family history of NTD and the other with history of DS demonstrated familial link between the two birth disorders [9]. However, this was not replicated in another study [10]. More uncommon than the occurrence of NTD and DS in the same family is their occurrence in the same individual. To the best of our information one case of a male child affected with both NTD and DS has been reported so far [11]. We report in this communication another case of a child affected with NTD and DS. In the reported case, the mother and the child had abnormal levels of folate metabolites and DNA hypomethylation in lymphocytes [11]. In the present communication we report common polymorphisms in genes encoding enzymes of the folate-dependent and independent pathways in the mother of the affected female child.
Subjects and Methods
The case was the second born female child of biologically unrelated parents of Asian Indian origin belonging to North India. Her condition was diagnosed by one of us (SJK) at his Pediatric Surgery unit in Ishayu Pediatric Specialty Clinic, Mumbai. The mother was 39 years old at childbirth, two years before her inclusion in the study. The infant was delivered full term by Caesarian section. Ultrasound of the spine revealed a tethered cord and a lumbar myelomengocoele with cauda equina roots in the thecal sac, diagnostic of NTD. Chromosomal analysis of peripheral blood lymphocytes stimulated with phytohaemagglutinin revealed trisomy of chromosome 21. The karyotype was diagnostic of DS. The 9-year older sibling of the case was a normal female child.
The mother was a hypertensive patient since her second pregnancy that ended in miscarriage. She was on medication with alpha-methyl dopa throughout her third pregnancy that ended in the birth of the affected offspring. Presently the mother is on medication with calcium channel antagonist namely amlodipine, besides calcium and iron supplements. After being explained the purpose of the study, she gave her written informed consent to participate. A sample of blood was withdrawn from the ante-cubital vein with minimum stasis. Genomic DNA was extracted from peripheral blood leucocytes for documenting DNA polymorphisms.
The DNA polymorphisms that were determined in the mother by previously described methods were as follows: i) methylene tetrahydrofolate reductase gene (MTHFR) 677C>T [12]; ii) methionine synthase gene (MTR) 2759A>G [13]; iii) betaine homocysteine methyl transferase gene (BHMT)742G>A [14]; iv) thymidylate synthase gene enhancer region (TSER) – three or two copies of 28-bp tandem repeats [15]; and v) TS gene, 6-bp deletion in 3’ untranslated region (UTR) [16]. The genotype and minor allele frequencies at these loci were determined in a sample of female subjects enrolled in a previous study, to compare the combined genotype of the case mother with this group. The details of enrolment are described elsewhere [17]. Briefly, this group consisted of 114 females, 57 with angiographically diagnosed coronary artery disease (CAD) and 57 age-matched asymptomatic controls from the general population [17]. None of the women with CAD had history of children born with NTD or DS as documented in the hospital case records. In case of control women this information was not available.
Results and Discussion
The body mass index of the case-mother computed from her height and weight measurements was 25.5 Kg/M2. She reported being a life long vegetarian. Vegetarianism spanned across generations in her family. Her daily intake of butter, ghee (clarified butter) and vegetable oils were in the first, second and third quintiles respectively of daily intake of the control group of women. She may probably have a restricted dietary intake of choline that is present more in animal than in vegetable fats. Choline is a precursor of betaine, the substrate of BHMT enzyme. Her daily intake of vitamin B12 (present mainly in foods of animal origin) assessed from standard 24-hour recall and food frequency questionnaire was 0.69 µg, well below the recommended daily allowance of 2.4 µg [18]. The case-mother was found to have a single copy of variant allele (heterozygous) for MTHFR 677C>T and MTR 2756A>G single nucleotide polymorphisms (SNPs), TSER repeat polymorphism and TS 6-bp deletion polymorphism, but two copies of the variant allele (homozygous variant) for BHMT 742G>A SNP. The genotype distribution and minor allele frequencies of these polymorphisms in the female population is given in Table 1. The number of female control subjects with the same combination of genotypes at multiple loci as in the case-mother was counted (Table 2). Unlike in the case-mother, the BHMT 742AA genotype was either absent or present in a small number of control women in combination of genotypes at five, four or three polymorphic loci (Table 2).
It is difficult to identify maternal risk factor(s) for NTD and DS-affected offspring in the present case. Although the variant allele frequency of the BHMT 742G>A SNP was present in 25% and BHMT 742AA genotype in 6% of the female controls (Table 1), the 742AA genotype in combination with other folate genotypes at three, four or five loci in the case-mother was present in none (0/110) to 2.7% (3/110) of the controls (Table 2). This may suggest that among folate gene polymorphisms documented, BHMT 742AA genotype is likely to be associated with increased risk for the case born with NTD and DS. Although the effect of ‘A’ allele on BHMT activity is not established, one study suggested an increased maternal risk for NTD in its presence [19]. Dietary deficiency of choline was reported to increase the maternal risk of having a baby with NTD [20]. A life-long vegetarian diet of the case-mother with a low intake of vitamin B12 – dietary co-factor of MTR of folate-dependent pathway of remethylation of homocysteine, and a presumed restricted intake of choline with possible altered BHMT activity due to the presence of SNP in two copies may impact upon the folate-independent pathway. It may be speculated that these could compromise remethylation of homocysteine and impair biological methylation thereby enhancing maternal risk for NTD and DS.
References
1. Larry JM, Edmonds ZD. Prevalence of spina bifida at birth – United States 1983-1990. A comparison of two surveillance systems. Mortality Morbidity Weekly Report 1996; 45: 15-26
2. Committee of Genetics. American Academy of Pediatrics. Folic acid for the prevention of neural tube defects. Pediatrics 1999; 104: 325-7
3. Verma IC. Burden of genetic disorders in India. Indian J Pediatrics 2000; 67: 893-8
4. Kulkarni ML, Mathew MAQ, Ramchandran B. High incidence of neural tube defects in south India. Lancet 1987: i: 260
5. Shin M, Besser LM, Kucik JE, Lu C, Siffel C, Correa A; Congenital Anomaly Multistate Prevalence and Survival Collaborative. Prevalence of Down syndrome among children and adolescents in 10 regions of the United States. Pediatrics 2009; 124: 1565-71
6. Verma IC, Bijarnia S. The burden of genetic disorders in India and a framework for community control. Community Genetics 2002; 5: 192-6.
7. Put NM van der, Thomas CM, Esikes TK et al. Altered folate and vitamin B12 metabolism in families with spina bifida offspring. Quart J Med 1997; 90: 505-10
8. Hobbs CA, Sherman SI, Ping Y et al. Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome. Am J Hum Genet 2000; 67: 623-30
9. Barkai G, Arbuzova S, Berkenstadt M, Hiefetz S, Cuckle H. Frequency of Down’s syndrome and neural tube defects in the same family. Lancet 2003; 361: 1331-5
10. Amorim MR, Castilla EE, Orioli IM. Is there a familial lin between Down’s syndrome and neural tube defects? Population and familial survey. Brit J Med 2004; 84: 328-
11. Al-Gazali LI, Padmanabhan R, Melnyk S, Yi P, Pogribny IP, Bakir M, Hamid ZA, Abdulrazzaq Y, Dawodu A, James SJ. Abnormal folate metabolism and genetic polymorphism of the folate pathway in a child with Down syndrome and neural tube defect. Am J Med Genet 2001; 103: 128-32
12. Mukherjee M, Joshi S, Bagadi S, Dalvi M, Rao A, Shetty KR. A low prevalence of the C677T mutation in the methylenetetrahydrofolate reductase gene in Asian Indians. Clin Genet 2002;61:155-9.
13. Laraqui A, Allami A, Carrie A et al. Relationship between plasma homocysteine, gene polymorphism of homocysteine metabolism related enzyme and the angiographically proven coronary artery disease. European Journal of Internal Medicine. 2007; 18:474-483
14. Ananth CV, Elsasser DA, Kinzler WL et al. Polymorphisms in methionine synthase reductase and betaine-homocysteine S-methyltransferase genes: Risk of placental abruption. Molecular Genetics and Metabolism.2007;91:104-110
15. Horie N, Aiba H, Ogura K, Hojo H, Takeishi K. Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5’ terminal regulatory region of the human gene thymidylate synthase. Cell Structure and Function. 1995; 20: 191-197
16. Ulrich CM, Bigler J, Velicer C, Greence E, Farin F Potter J. Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiology, Biomarker and Prevention. 2000;9:1381-1385
17. Mukherjee M, Brouilette S, Stevens S, Shetty KR, Samani NJ. Association of shorter telomeres with coronary artery disease in Indian subjects. Heart 2009; 95: 669-73
18. USDA National Nutrient Database for Standard Reference, Release 21. In: US Department of Agriculture ARS, ed. 2008
19. Boyles AL, Billups AV, Deak KL, Siegel DG, Mehltretter L, Slifer SH, Bassuk AG, Kessler JA, Reed MC, Nijhout HF, George TM, Enterline DS, Gilbert JR, Speer MC; NTD Collaborative Group. Neural tube defects and folate pathway genes: family-based association tests of gene-gene and gene-environment interactions. Environ Health Perspect 2006;114:1547-52.
20. Shaw GM, Carmichael SL, Yang W, Selvin S, Schaeffer DM. Periconceptional dietary intake of choline and betaine and neural tube defects in offspring. Am J Epidemiol 2004; 160: 102-9.
Source(s) of Funding
The study was supported by an institutional grant (ICT, Mumbai) from World Bank’s Technical Quality Improvement Program (TEQIP) ‘Service to Society’ scheme and Baun Foundation Trust (BFT), Mumbai at Cumballa Hill Hospital and Heart Institute. BFT supported PRS.
Competing Interests
None to declare
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