Urinary Organic Acid Profiles in Children with Developmental Delay According to MTHFR C677T and A1298C Compound Genotypes: A Quantitative Analysis

Urinary Organic Acid Profiles in Children with Developmental Delay According to MTHFR C677T and A1298C Compound Genotypes: A Reanalysis with Complete Data (N=225)

Ilgu Kim, MD Suavel Clinic, Daegu, South Korea

Abstract

Objective: To investigate the association between MTHFR C677T and A1298C polymorphisms and urinary organic acid markers (Methylmalonate, Isocitrate, HVA, Hippurate, Orotate), thereby elucidating the genetic basis of organic acid metabolic abnormalities in children with developmental delay.

Subjects and Methods: A total of 225 children who underwent both MTHFR C677T/A1298C genotyping and urinary organic acid testing at Suavel Clinic were included. Organic acid levels were reclassified into a three-tier system — decreased (−1) / normal (0) / elevated (+1) — by directly comparing measured values (value) against reference ranges (ref_range). Pearson correlation analysis, Kruskal-Wallis test, and Mann-Whitney U test were performed.

Results: A statistically significant positive correlation was identified between A1298C genotype and HVA score (Pearson r = 0.206, p = 0.002; Kruskal-Wallis H = 16.88, p = 0.0002). Mean HVA scores increased stepwise from AA → AC → CC genotypes: 0.04 → 0.09 → 0.57. C677T showed no significant correlation with any of the markers. Isocitrate elevation was the most frequent finding at 52.2%, but showed no significant association with MTHFR variants. Orotate elevation was observed in 38.4% of subjects.

Conclusion: The A1298C variant, particularly the homozygous (CC) genotype, is significantly associated with elevated urinary HVA, suggesting involvement in dopamine metabolic dysregulation. These findings may serve as a foundation for targeted supplementation therapy in affected children.

Keywords: MTHFR, compound heterozygote, C677T, A1298C, urinary organic acids, HVA, orotate, developmental delay

Introduction

Polymorphisms in the MTHFR (methylenetetrahydrofolate reductase) gene play a central role in the folate metabolic pathway and have been consistently implicated in neurodevelopmental disorders. Among these, the C677T and A1298C variants — whether occurring independently or as compound heterozygotes — may exert influence on the methylation cycle and neurotransmitter biosynthesis.

Urinary organic acid testing offers a non-invasive means of evaluating mitochondrial function, intestinal microbial metabolism, and neurotransmitter metabolism, and is widely utilized in functional medicine assessments of children with developmental delay. Nevertheless, quantitative clinical data from Korean cohorts on the relationship between MTHFR polymorphisms and organic acid markers remain limited.

The present study addresses the methodological limitations of a prior analysis (n = 148; parsing of 5–6 markers) by applying an upgraded organic acid parser (complete parsing of 36 markers, with Orotate values recovered) and a three-tier reclassification method, yielding a complete dataset of 225 subjects for reanalysis.

Methods

Study Population

Two hundred and twenty-five children who underwent both MTHFR C677T/A1298C genotyping and urinary organic acid testing at Suavel Clinic between 2022 and 2025 were included. Five organic acid markers were analyzed: Methylmalonate, Isocitrate, HVA (Homovanillic acid), Hippurate, and Orotate.

The increase in the overlapping dataset compared to the prior analysis (n = 148) was primarily attributable to improvements in the organic acid parser — specifically, expansion from parsing 5–6 markers to complete parsing of 36 markers, and recovery of Orotate measured values.

Classification Method

In the prior analysis, a binary classification of normal (0) / abnormal (1) was applied based on the testing laboratory's "borderline" designations; however, inaccuracies in that classification scheme were subsequently identified. In the present study, each organic acid level was reclassified into a three-tier system — decreased (−1) / normal (0) / elevated (+1) — by directly comparing the measured value against the reference range.

Statistical Analysis

The association between MTHFR genotype and organic acid scores was evaluated using Pearson correlation analysis. Between-group comparisons by genotype were performed using the Kruskal-Wallis test, followed by post-hoc Mann-Whitney U tests. Statistical significance was set at p < 0.05.

Results

Distribution of Reclassified Organic Acid Markers

Following three-tier reclassification, Isocitrate elevation was the most frequent finding at 52.2%, followed by Orotate (38.4%), Hippurate (10.3%), HVA (9.8%), and Methylmalonate (7.6%).

Association Between A1298C and HVA

A statistically significant positive correlation was found between A1298C genotype and HVA score (Pearson r = 0.206, p = 0.002). The Kruskal-Wallis test likewise demonstrated significant between-group differences (H = 16.88, p = 0.0002), with mean HVA scores increasing in a stepwise fashion from AA → AC → CC genotypes. Mann-Whitney U post-hoc analysis confirmed a significant between-group difference (p = 0.035).

C677T and Organic Acid Markers

The C677T genotype showed no significant correlation with any of the organic acid markers analyzed — Methylmalonate, Isocitrate, HVA, Hippurate, or Orotate (all p > 0.1).

Other Markers

Hippurate and Methylmalonate showed no significant association with either MTHFR variant. Orotate elevation was observed in 38.4% of subjects; however, no statistically significant correlation with MTHFR variants was identified at the group level.

Discussion

The principal finding of this study is that the A1298C variant — particularly the homozygous (CC) genotype — is significantly associated with elevated urinary HVA. The A1298C variant is located in the regulatory domain of the MTHFR enzyme and influences the synthesis of BH4 (tetrahydrobiopterin), an essential cofactor for dopamine and serotonin biosynthesis. As the primary metabolite of dopamine, elevated HVA may reflect increased dopamine turnover or metabolic imbalance.

The absence of significant associations for C677T across all markers is likely attributable to its distinct functional role within the methylation cycle, which differs from that of A1298C.

Methodological Improvements and Differences from Prior Results: The previously reported finding of "90% Orotate abnormality" was determined to have arisen from incomplete classification by the earlier parser. With the application of the three-tier reclassification method based on measured values, the Orotate elevation rate has been corrected to 38.4%. Furthermore, the expansion of the analytical cohort from 148 to 225 subjects improved statistical power.

The high prevalence of Isocitrate elevation (52.2%) may be related to mitochondrial TCA cycle dysfunction or oxidative stress; however, a direct association with MTHFR variants was not demonstrated in the present study and warrants further investigation.

Limitations of this study include its single-center, retrospective design and the small sample size of the CC genotype subgroup (n = 4). Validation through multicenter prospective studies is warranted.

Conclusion

The A1298C variant demonstrates a significant positive correlation with elevated urinary HVA, with a particularly pronounced effect in the homozygous (CC) genotype. These findings suggest that the A1298C variant may influence dopamine metabolism and may provide a basis for targeted therapeutic interventions — such as BH4 supplementation — in children with developmental delay. The C677T variant showed no significant association with the organic acid markers analyzed. This study highlights the importance of complete marker parsing and measured-value-based reclassification in urinary organic acid analysis, and corrects the methodological errors identified in the prior analysis.

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Author: Ilgu Kim, MD Suavel Clinic, Daegu, South Korea