Birth defects represent a significant challenge in the field of global public health. According to data from the World Health Organization (WHO)[1], an estimated 8 million newborns worldwide are born with birth defects each year. In the Southeast Asia region, birth defects rank as the fourth most common cause of neonatal deaths, accounting for 12% of all neonatal mortality. Among the contributing factors, folic acid deficiency is one of the primary preventable causes. These defects not only endanger individual health but also impose a heavy burden on healthcare systems and society.

This issue is particularly prominent in China, where the incidence of neonatal birth defects is relatively high. Nationally, approximately 1 to 1.2 million babies are born with birth defects each year, equating to one new case every 30 seconds. Such a high incidence underscores the urgency of implementing effective preventive measures and targeted interventions.

Abnormalities in folic acid metabolism are a key factor leading to birth defects, as they disrupt DNA synthesis and methylation processes. These disturbances are not only associated with congenital diseases but also linked to certain cancers. Specifically, folic acid deficiency can lead to neural tube defects, congenital heart disease, cleft lip and palate, and other abnormalities[2].

Methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in the folic acid metabolic pathway, plays a central role in this process. Mutations in the MTHFR gene can impair folic acid metabolism, increasing the risk of adverse pregnancy outcomes such as miscarriage and fetal malformations. As a result, MTHFR testing has become an essential component of prenatal screening[3].

This project is dedicated to developing faster, more cost-effective, and convenient diagnostic methods for folic acid testing and formulating personalized intervention plans for different populations. Through in-depth research on folic acid metabolism and related genes, we aim to reduce the incidence of birth defects associated with folic acid deficiency and safeguard maternal and child health.

Every year, approximately 8 million newborns worldwide suffer from birth defects, accounting for 6% of all births. Birth defects are one of the leading causes of infant and child mortality, and survivors face a higher risk of long-term disabilities. The increase in birth defects poses severe challenges in multiple areas. As a populous country, China sees about 900,000 new cases of birth defects each year. For instance, treating a child with congenital heart disease costs an average of 100,000 yuan, while the economic burden of a new case of Down syndrome is around 450,000 yuan. This places a heavy financial strain on families and society, exacerbates the burden on the healthcare system, and can constrain economic growth due to reduced labor force.

In terms of population quality, children with birth defects often have limited labor capacity when they grow up. Some genetic factors leading to birth defects also increase the risk of illness in subsequent generations, affecting overall population quality. Folic acid deficiency is a major cause of birth defects. According to surveys, folic acid deficiency among women of childbearing age is very common globally. If folic acid metabolism abnormalities in pregnant women could be detected efficiently and accurately, and timely intervention provided, it would effectively reduce the number of birth defects.

Current detection technologies have significant limitations. Traditional screening methods such as serum folate and vitamin B12 level measurements can only reflect short-term folate nutritional status and fail to accurately demonstrate individual differences in folate metabolism capacity. Moreover, the testing process remains cumbersome and time-consuming. Some methods require specialized laboratory equipment and technical personnel, involving complex procedures and prolonged detection periods that typically take days or even weeks to produce results. This proves inconvenient for childbearing-age women needing timely information about their folate metabolism status.

Additionally, existing screening methods incur high cost and reliance on large instruments. While advanced detection technologies like next-generation sequencing (NGS)-based approaches demonstrate high accuracy, their expensive testing costs and long time to wait before getting results hinder widespread application in large-scale population screening. Some modern hospital laboratories in China offer MTHFR testing (e.g., for the C677T variant) with faster turnaround times, sometimes within the same day or a few working days 20. However, these are not universally accessible or may still involve centralized laboratory processing and highly rely on large and professional instruments. Furthermore, most current screening methods merely provide binary results regarding folate metabolism abnormalities without offering personalized folate supplementation guidance or tailored intervention plans, failing to meet the individualized needs of different women.

Therefore, developing an efficient, user-friendly, and cost-effective screening method for folate metabolism abnormalities holds significant implications for addressing population health challenges.

As showed in fig.7, two different FQ probes are designed to recognize the mutation of MTHRF gene with RNase H II protein. When the probe completely bond with template, the RNase H II was triggered and then catalyzed the probe. Then the polymerase associated with the primer, inducing 5’-3’ extension and strand displacement. Thus FAM and BHQ separated, leading to the formation of fluorescent signal.

The probes are added in one tube with sample, and then keep a 65℃ reaction in real-time PCR (qPCR) instrument to monitoring fluorescent data. The wild-type gene shows high fluorescent intensity with wild-type probe and low fluorescent of mutant probe. The mutant sample shows a contrary outcome with wild-type gene, while the heterozygous sample shows high fluorescent signal with both wild-type probe and mutant probe. The result improve the high sensitivity and specificity of our strategy.

We designed two probes based on base complementarity and SNP. Initially, we used single probes to detect and verify wild-type and mutant genes. The experimental results showed that the wild-type probe was highly sensitive to wild-type gene samples and heterozygous samples, but almost no fluorescence changes were detected in mutant samples. Conversely, the mutant probe was highly sensitive to mutant and heterozygous genes, but did not produce fluorescence signals with wild-type genes. This was further confirmed by plasmid experiments. Subsequently, we attempted to add both wild-type and mutant probes to the detection system to achieve single-tube, rapid detection of the three genotypes. The results demonstrated that combining wild-type and mutant probes could effectively distinguish between the three genotypes.

On this basis, we further tested our system with actual blood samples to explore its potential in practical applications. The experimental results showed that our detection system was completely consistent with the results of second-generation sequencing in 50 actual samples. Based on this scheme, we plan to develop a faster, simpler, and lower-cost screening method and design a POCT to ensure that our detection method can be widely popularized and applied. We further improved our strategy with isothermal Amplification and designed Colloidal Gold Immunoassay Test Strip to achieve the implementation of folic acid metabolism screening using portable and miniaturize instruments to facilitate the promotion of eugenics and the prevention of birth defects in less developed areas. Our project enables rapid folate metabolism screening anywhere, anytime, without technical barriers or complexities.

Currently, most hospitals offer genetic screening for folate metabolism. However, its high cost (200–500 RMB per sample) and prolonged turnaround time limit widespread adoption. In contrast, our technology reduces costs by analyzing different results of quantitative PCR, making it suitable for primary healthcare settings. Dual-probe single-tube detection enables simultaneous differentiation of homozygous wild-type, heterozygous, and homozygous mutants, with per-sample costs as low as 3 RMB.

Our project is advancing toward automation and multi-omics integration. The POCT equipment designed for the project integrates the whole process of “blood collection - lysis - amplification - interpretation”, and the testing time is shortened to 1 hour. Our project will construct a more rapid, economical and convenient folate metabolism diagnostics technique to play a more central role in birth defect prevention worldwide.

This comparative analysis highlights the unique combination of extreme low cost, rapidity, and potential for decentralization via POCT that defines the innovative edge of this project.

In recent years, the Chinese government has placed significant emphasis on the comprehensive prevention and control of birth defects, continuously refining its policy framework to provide robust support for prenatal testing and folic acid supplementation among pregnant women. According to Wise Guy Reports' market overview of folic acid metabolism genetic testing, the market size for such tests was estimated at USD 1.71 billion in 2023. The industry is projected to grow from USD 1.82 billion in 2024 to USD 3.05 billion by 2032, with a compound annual growth rate (CAGR) of approximately 6.65% during the forecast period (2025–2032).

Policy remains one of the most critical factors influencing the pharmaceutical industry. With increasing national attention to neonatal defects and rising demand among pregnant women for prenatal testing, China has implemented a series of policies since 2017 to advance the development of prenatal testing and related pharmaceuticals. Key examples include:

July 2017: The Basic Public Health Service Standards (Third Edition) explicitly incorporated maternal health management into the national basic public health service program, mandating grassroots medical institutions to provide free folic acid distribution and follow-up services for pregnant women.

January 2021: The revised Technical Management Measures for Prenatal Screening and Diagnosis promoted non-invasive prenatal testing (NIPT) as a first-line screening method for chromosomal abnormalities such as Down syndrome.

August 2023: The National Comprehensive Prevention and Control Plan for Birth Defects (2023 Revision) further stipulated that the folic acid supplementation rate before and during early pregnancy must exceed 90% by 2025. It also integrated folic acid metabolism genetic testing (e.g., MTHFR C677T) into pre-pregnancy优生health check-up programs in select regions.

These measures collectively underscore China's strong policy support for the development of prenatal testing reagents, indicating a promising growth trajectory for the prenatal testing and folic acid supplementation market. Such initiatives also provide encouragement and assistance for the advancement of our project.

[1] “World Birth Defects Day: Many birth defects, one voice.” Accessed: May 11, 2025. [Online]. Available: https://www.who.int/southeastasia/news/detail/02-03-2023-world-birth-defects-day-many-birth-defects-one-voice
[2] F. H. Nazki, A. S. Sameer, and B. A. Ganaie, “Folate: Metabolism, genes, polymorphisms and the associated diseases,” Gene, vol. 533, no. 1, pp. 11–20, Jan. 2014, doi: 10.1016/j.gene.2013.09.063.
[3] S.-C. Liew and E. D. Gupta, “Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: Epidemiology, metabolism and the associated diseases,” Eur. J. Med. Genet., vol. 58, no. 1, pp. 1–10, Jan. 2015, doi: 10.1016/j.ejmg.2014.10.004.