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Journal of Molecular and Clinical Medicine  2018, Vol. 1 Issue (2): 119-126    DOI: 10.31083/j.jmcm.2018.02.010
Research article Previous articles |
Elucidation of Folate-Mediated Cascades in the Developing Neural Tube: Congenital Malformations Induced by Methyltransferase Inhibition
Robert M Cabrera1, *(), Bogdan J1, *(), Wlodarczy k1, Richard H. Finnell1, *()
1 Departments of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
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Folic acid supplementation has been shown to significantly reduce both the occurrence and the recurrence of neural tube defects (NTDs) in human populations, yet the underlying mechanisms for reducing the risk of NTDs continue to be debated. This study examined genetic background and select folate metabolites as possible modifiers that may influence NTD risk. Specifically, several folate cycle and methylation metabolites were examined for their ability to reduce the occurrence of NTDs in two congenic mouse strains carrying targeted disruption of the folate receptor 1 (Folr1) gene. SWV-Folr1$^{tm1Fnn}$ and LM/Bc-Folr1$^{tm1Fnn}$ mice were provided with folate or several folate pathway metabolites, or combinations thereof, to determine the ability of these compounds to rescue nullizygous embryos from lethality and NTDs. Results demonstrated that SWV-Folr1$^{tm1Fnn}$ and LM/Bc-Folr1$^{tm1Fnn}$ mice exhibit different dose responses to folinic acid (5-formyl-tetrahydrofolate) supplementation; however, treating dams throughout gestation with downstream metabolites indicated that only folates rescued Folr1 nullizygous embryos from lethality and NTDs. Chemical inhibitors of folate metabolism were used to further elucidate essential enzymatic and biochemical metabolites. These data demonstrate that the inhibition of S-adenosyl-L-homocysteine hydrolase (AHCY) or selective inhibition of folate responsive isoprenylcysteine carboxylmethyltransferase (ICMT) results in embryo toxicity and fetuses with anterior NTDs. These data indicate that genetic background modifies NTD penetrance in folate-supplemented Folr1$^{tm1Fnn}$ mutants, while downstream metabolites of folate metabolism are not capable of rescuing Folr1$^{tm1Fnn}$ mutants. Moreover, these findings support the hypothesis that the methylation cycle and post-translational methylation of key signaling proteins such as Ras, by ICMT are essential to neural tube closure.

Key words:  Folic acid      CAAX      Neplanocin A      3-deazaneplanocin A      3-deazaadenosine      Periodate-oxidized adenosine      ICMT      Methyltransferase      Neural tube defects     
Submitted:  07 March 2018      Revised:  19 March 2018      Accepted:  21 March 2018      Published:  20 April 2018     
*Corresponding Author(s):  Robert M Cabrera,Bogdan J,Richard H. Finnell     E-mail:;;

Cite this article: 

Robert M Cabrera, Bogdan J, Wlodarczy k, Richard H. Finnell. Elucidation of Folate-Mediated Cascades in the Developing Neural Tube: Congenital Malformations Induced by Methyltransferase Inhibition. Journal of Molecular and Clinical Medicine, 2018, 1(2): 119-126.

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Fig. 1.  Folate biochemistry and the methionine cycle.
Substrates: tetrahydrofolate (THF); 5, 10-methylene-tetrahydrofolic acid (5, 10-CH$_{2}$-THF); 5-methyl-tetrahydrofolate (5-CH$_{3}$-THF); S-adenosylmethionine (SAM); S-adenosylhomocysteine (SAH); homocysteine (Hcy)
Enzymes: 1. dihydrofolate reductase (DHFR); 2. serine hydroxymethyltransferases (SHMT1/SHMT2); 3. Methylene-tetrahydrofolate reductase (MTHFR); 4. methionine synthase (MS, MTR); 5. methionine adenosyltransferase (MAT); 6a. farnesyltransferase, 6b. CAAX prenyl protease 2 (Ras converting enzyme type 1, RCE1), 6c. isoprenylcysteine carboxyl methyltransferase (ICMT); 7. S-adenosylhomocysteine hydrolase (AHCY).
In the current study, small molecules were used to inhibit methytransferases globally (7, AHCY) or specifically (6c, ICMT). The methylation of CAAX proteins (e.g. Ras and Rho GTPases) utilizes 6a catalyzed lipidation (addition of prenylated lipids), 6b catalyzed endoproteolytic cleavage of the AAX amino acids, and 6c catalyzed carboxyl-methylation of the isoprenylcysteine.

Fig. 2.  Linkage of the Folr1 and Tyrosinase (SWV albinism) loci.
Due to the frequent occurrence of blonde coat color with Folr1 genotype during backcrossing onto the SWV strain, genomic analysis was performed. The 14.5 Mbase proximity is consistent with linked traits that can frequently segregate with mutant loci.

Fig. 3.  Rescue response frequencies for Folr1.
Response frequencies are plotted for SFA and indicated that both the SWV-Folr1$^{tm1Fnn}$ and LM/Bc-Folr1$^{tm1Fnn}$ mice have folate-responsive NTDs. Genetic background strain is shown as a major modifier of NTD penetrance. The response to supplementation also differed by maternal genotype.

Fig. 4.  Tested folate metabolites.
Testing strategy for methyl cycle metabolites and nucleosides are shown. All treatments were calculated as approximately 88 mmole/Kg equivalents of folinic acid (SFA). There were no nullizygous fetuses from dams receiving either the negative control (water) or individual folate metabolites. Only SFA resulted in any nullizygous fetuses surviving to term.

Table 1  Developmental toxicity due to inhibition of AHCY.
Treatment Total Total % Unaffected % Affected
Litters NTD Affected Litters Implants Normal Dead NTDs Resorptions
DZA 40mg/Kg 7 4 77 53 1 12 11 68.8% 31.2%
DZA 80mg/Kg 3 1 41 6 0 6 29 14.6% 85.4%
DZNep 5mg/Kg 14 1 171 148 7 3 13 86.5% 13.5%
DZNep 7.5mg/Kg 9 1 107 47 1 1 58 43.9% 56.1%
DZNep 10mg/Kg 6 0 69 13 0 0 56 18.8% 81.2%
AdOx 40mg/Kg 14 1 183 164 2 1 16 89.6% 10.4%
AdOx 80mg/Kg 3 0 38 36 0 0 2 94.7% 5.3%
Nep A 4mg/Kg 7 0 84 79 2 0 3 94.0% 6.0%
Nep A 8mg/Kg 6 0 78 74 2 0 2 94.9% 5.1%
Fig. 5.  Inhibitors of AHCY.
Summary of maternal and developmental toxicity of adenosine analogs and AHCY inhibitors. The order of teratogenicity was DZA $>$ DZNep $>$ AdOx* $>$ NepA. The maternally toxic dosage for NepA was reached without a significant increase in developmental toxicity. *There was one fetus with an NTD and midline facial cleft at maternally toxic dosages for AdOx, 4 of 18 moms died at 40 mg/Kg AdOx, $>$ 10% resorption. Both DZA and DZNep produced fetuses from multiple litters with NTDs.

Fig. 6.  Congenital Malformations due to ICMT inhibition.
The inhibition of ICMT via N-Acetyl-S-geranylgeranyl-L-cysteine (AGGC) administration at E8.5 resulted in three litters with congenital malformation including exencephaly and gastroschisis. There were no defects in animals exposed to N-acetyl-S-geranyl-L-cysteine (AGC).

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