Pseudo-Methyl Malonic Acidemia in Indian Babies Secondary to Maternal Vitamin B12 Deficiency

Background: Organic acidemias (OAs) are inherited disorders of intermediary metabolism caused by deficiencies in specific enzymes or transport proteins. Methylmalonic acidemia (MMA) is a prevalent type of OA that arises from a deficiency in methylmalonyl-CoA mutase. Newborn screening (NBS) is crucial for the early detection and management of MMA and other metabolic disorders. However, external factors, such as maternal vitamin B12 deficiency, can lead to false-positive results and impact the accuracy of NBS for MMA.

Aim: This study aimed to assess the accuracy of NBS for detecting MMA and determine the prevalence of false-positive cases caused by external factors, such as maternal vitamin B12 deficiency.

Methods: This multicentric study conducted NBS for MMA on 1,55,420 newborns from multiple hospitals in the Cloudnine Hospital Network in India. The samples were collected from babies born between 19 January 2007 and 19 September 2023. The study obtained dried blood spots from infants 36 hours after birth and analyzed the samples using tandem mass spectrometry. Ethical clearance and informed consent were obtained from parents/guardians, and confidentiality and data protection measures were observed.

Results: A total of 639 samples (0.41%) showed elevated propionyl-carnitine and methylmalonic acid levels, initially suggesting MMA. However, further evaluation revealed that these abnormalities were linked to maternal vitamin B12 deficiency, resulting in false-positive results. The mothers of the affected infants, after confirmation of low vitamin B12 levels, received vitamin B12 injections, and a follow-up analysis on the infants after 3 weeks showed that the methylmalonic acid levels in all 637 samples had returned to normal. 2 samples showed no maternal vitamin B12 deficiencies, indicating true Methylmalonic acidemia.

Conclusions: This study highlights the importance of considering external factors, such as maternal health and dietary influences, in interpreting NBS results for MMA including maternal vitamin B12 deficiency which can lead to false-positive results, impacting the accuracy of NBS. Therefore, a holistic approach to NBS is essential, incorporating both, the possibility of inborn errors of metabolism in the baby, as well as external influences such as maternal health. In addition, undiagnosed vitamin B12 deficiency in mothers could lead to unrecognized neurological problems and morbidities in children at a later age due to pseudo-MMA.

Organic acidemias (OAs), also known as organic acidurias, constitute a group of inherited disorders of intermediary metabolism. These conditions primarily arise from deficiencies of specific enzymes or transport proteins within metabolic pathways involved in breaking down amino acids, carbohydrates, or lipids (Ramsay et al., 2018). Among OAs, propionic acidemia, methylmalonic acidemia (MMA), isovaleric acidemia, and maple syrup urine disease (MSUD) are categorized as the most prevalent inborn errors of metabolism (IEMs) related to the catabolism of branched-chain amino acids (BCAA). These conditions are commonly referred to as “classical OAs” (Rossi et al., 2020). Anecdotal evidence in India suggests that their incidence is high in India, though reported cases are patchy.

Newborn screening (NBS) is a crucial public health program aimed at enhancing outcomes for children with treatable disorders that may not be detected during routine care. Several disorders identified through NBS are IEMs, which encompass conditions such as MMA, and disorders related to cobalamin metabolism (Held et al., 2022). Methyl malonic acidemia is biochemically identified by the build-up of methylmalonic acid in various bodily fluids and tissues and is caused by a deficiency of the enzyme methylmalonyl-coenzyme A (CoA) mutase (Almasi et al., 2019). There are two primary forms of MMA: isolated MMA and combined MMA. The isolated form may arise from a complete or partial deficiency of methylmalonyl-CoA mutase.This deficiency can be classified into two enzymatic subtypes: mut0 and mut−, representing complete and partial enzyme deficiency, respectively (Almasi et al., 2019).Combined MMA occurs when MMA is accompanied by homocystinuria and/or homocystinemia (Sloan et al., 2021). In newborns, MMA is detected through NBS when elevated propionylcarnitine (C3) levels or propionylcarnitine/acetylcarnitine (C3/C2) ratios are observed in the blood (Peng et al., 2019).

The clinical severity of MMA can vary, from being a benign condition to a potentially fatal neonatal disease. The onset of MMA can occur at different stages, ranging from the neonatal period to adulthood (Held et al., 2022). The clinical features associated with MMA are diverse and can vary depending on the specific subtype of the disorder. Common clinical manifestations include anorexia, failure to thrive, hypotonia, developmental delay, progressive renal failure, functional immune impairment, optic nerve atrophy, and hematologic abnormalities (Barahate et al., 2023). The presentation of these symptoms can be complex and multifaceted, making the diagnosis and management of MMA challenging. 

The worldwide pooled prevalence of MMA has been reported to be 1.14 per 100,000 newborns (95% confidence interval [CI]=0.99–1.29). In patients clinically suspected of MMA, the pooled prevalence was 652.11 per 100,000 individuals (95% CI: 544.14–760.07) (Jin et al., 2022). The pooled prevalence of MMA in Asia and Africa is reported to be higher than that of other global regions (Jin et al., 2022). In a study conducted to assess the prevalence of MMA in Asian countries, a total of 293 cases of IEMs were identified out of 2105 examined cases. Among these, MMA was the most prevalent, accounting for 72 out of 293 cases (Shibata et al., 2018). This multicentric study conducted by the Cloudnine Hospital Network (CHN) aimed to evaluate the significance and cost-effectiveness of implementing a routine NBS program for metabolic screening, such as that for MMA, in India. While this practice is already established in various regions worldwide, its relevance and feasibility in the Indian context are yet to be established especially regarding maternal vitamin B12 deficiency.

Study design: This was a multicentric, prospective study involving NBS for detecting MMA in newborns from various CHN hospitals in South India. A total of 1,55,420 blood samples from newborns born between 19 January 2007 and 19 September 2023 were collected and analyzed. 

Ethics statement

This study received ethical clearance from the Ethical Committee and Institutional Review Board of the CHN. The samples were collected in congruence with the principles outlined in the Declaration of Helsinki and in adherence to the relevant national and international guidelines for ethical research involving human subjects. Written informed consent was obtained from all the parents or legal guardians of the newborns whose samples were included in the study. The parents/guardians were provided with detailed information about the study’s objectives, potential risks, and benefits. Confidentiality and data protection measures were strictly observed, and all the collected data were anonymized and securely stored. 

Sample collection

Dried blood spots (DBS) were collected from newborn babies at 36 hours of age using venipuncture methods as the blood was being drawn from the babies for other tests (including those for bilirubin levels and identifying blood group) as per the hospital policy. The venipuncture method involved using a winged steel needle (23 or 23 gauge) with an extension tube. The child was kept warm and immobilized by a parent or the phlebotomist. A transilluminator was used to locate veins and the skin was punctured 3–5 mm away from the vein. The blood was drawn slowly and steadily using a syringe or evacuated tube. A drop of the collected blood was placed on specialized filter paper and dried to obtain the DBS sample. To collect the blood via the heel-prick method, the baby's heel was gently warmed using a warm pack or a soft, warm, moist cloth at a temperature of up to 106°F (41°C) for 3–5 minutes. The collection site was then cleaned using an alcohol prep pad, followed by drying with a sterile gauze pad. Next, a small puncture was made on the heel with a sterile needle, and the first blood drop was gently wiped away using a sterile gauze pad. A larger blood drop was allowed to form, which was soaked up by a specialized filter paper used for the sampling. After this, the filter papers with the blood spots were carefully dried on a clean, flat, non-absorbent surface for a minimum of 3 hours. Throughout this process, the filter papers were kept away from direct sunlight and other sources of heat to ensure the stability and integrity of the samples. The samples were then analyzed by tandem mass spectrometry. 

Statistical analysis

All statistical analyses were performed using Microsoft 365 Excel. The data expressed on a qualitative scale were presented as the number and percentage of the sample. Results were considered statistically significant if the p-value was less than 0.05.

Testing

Out of the total 1,55,420 DBS collected and analyzed, slightly elevated levels of propionyl-carnitine (normal range=0.3–4.2 µmol/L) and methylmalonic acid (less than 1.1 µmol/L) were observed in 0.41% (639/1,55,420) of the samples. However, these elevated levels may be linked to maternal vitamin B12 deficiency, which is not indicative of MMA. Only 2 samples had elevated levels of propionyl-carnitine and methylmalonic acid levels with normal levels of maternal vitamin B12 – and further testing confirmed MMA in these 2 babies & these 2 babies are not discussed here.

Post-testing

Parents of infants with abnormal values of propionyl-carnitine and methylmalonic acid were provided with comprehensive counseling, and maternal vitamin B12 levels were meticulously assessed. In most cases, maternal B12 levels were low (typically 30–120 pmole/L, whereas normal B12 levels should be 150–800 pmole/L). To address the issue of low vitamin B12 levels, monthly vitamin B12 injections were administered intramuscularly to the mothers. Subsequently, when the NBS test was repeated after a span of four to 6 weeks for these infants, all 637 sample values were within the normal limits, signifying a positive response to the intervention.

Methylmalonic acidemia is a genetic condition that can have unfavorable outcomes (Barahate et al., 2023). Usually, the symptoms of MMA are not noticeable at birth or in the first few months of life; symptoms usually arise during the first year of life, when the child’s diet includes proteins other than those in breastmilk (Barahate et al., 2023; Saini et al., 2015). Although MMA was found to be the most prevalent IEM in Asia and Africa, a recent systematic review indicates that MMA is an ultra-rare disorder (Almasi et al., 2019; Shibata et al., 2018). However, none of these studies have specifically investigated maternal vitamin B12 levels. However, early recognition and appropriate medical intervention are essential for effectively managing the disorder and improving the overall quality of life for individuals affected by MMA. Timely and comprehensive treatment can help mitigate the impact of the condition and minimize potential complications (Malvagia et al., 2020). 

In this study, only 0.41% (637/1,55,420) of all the screened newborns had elevated propionyl-carnitine and methylmalonic acid levels. However, these were not indicative of MMA, but were linked to maternal vitamin B12 deficiency. It is known that maternal vitamin B12 deficiency can lead to slight elevations in propionyl-carnitine and methylmalonic acid, and cause homocystinuria in the newborns of mothers with vitamin B12 deficiency (Sloan et al., 2021). Maternal vitamin B12 deficiency can lead to varying presentations in infants from mild (detection of elevated propionyl carnitine and methylmalonic acid levels through NBS) to severe encephalopathy (Dror et al, 2008; Sloan et al., 2021; Rivada et al., 2022). Administering intramuscular vitamin B12 replacement therapy to normalize vitamin B12 serum concentrations can effectively reverse the metabolic abnormality (Sloan et al., 2021). Methylmalonic acid and homocysteine play crucial roles as intermediates in vitamin B12 and folate metabolism, and they serve as valuable supplementary biomarkers for diagnosing deficiencies in both vitamin B12 and folate (Torrez et al., 2021). In patients with vitamin B12 deficiency, both methylmalonic acid and homocysteine levels are elevated (Torrez et al., 2021). Therefore, when vitamin B12 concentrations are borderline, it is advisable to measure methylmalonic acid concentrations to aid in the diagnosis of a deficiency (Torrez et al., 2021). 

In this study, when the infants with elevated propionyl carnitine and methylmalonic acid levels were detected, the mothers were administered vitamin B12 injections after confirming the existence of maternal vitamin B12 deficiency. During the follow ups four weeks later, the children had neither elevated level of these two MMA biomarkers nor low vitamin B12 levels. This finding raises concerns about the high rates of “pseudo-MMA” that may be obtained in NBS studies, especially as elevated C3 levels in newborns, caused by maternal vitamin B12 deficiency, are a common cause of false positives during NBS for the detection of MMA (Rivada et al., 2022). In South Asian countries such as India and Nepal, “MMA-like diseases” or pseudo-MMAs resulting from vitamin B12 deficiency are widely recognized and may manifest themselves as various disorders such as infantile tremor syndrome (ITS), also known as “vitamin B12 deficiency syndrome” (Shibata et al., 2018; Gupta et al., 2022). Infact the Indian Journals still keep publishing papers in various forms even today. (Kesavan et al., 2023)

Moreover, it is well-established that small amounts of methylmalonic acid in the blood of normal, healthy infants during the first days of life are not uncommon due to the immaturity of the hepatic enzymes. As a result, inaccuracies in setting cut-off levels for methylmalonic acid during NBS using DBS could potentially lead to a higher number of false positives than anticipated (Malvagia et al., 2020). In a study by Pajares et al. (2023), it was shown that the inclusion of MMA, methyl citric acid (MCA), and homocysteine as secondary tests in NBS programs were successful in detecting acquired vitamin B12 deficiency and genetic defects. Furthermore, it reduced the false positive rates in detecting MMA; elevated homocysteine levels were the best marker for acquired vitamin B12 deficiency, whereas elevated MCA levels pointed to genetic defects (Pajares et al., 2023).High vitamin B12 levels in newborns are also linked to MMA pathophysiology and organ damage, serving as an independent risk factor for MMA symptom onset. Using serum vitamin B12 levels as an auxiliary index could aid in accurate MMA diagnosis alongside additional analyses in children clinically suspected of MMA (Du et al., 2023).

This study has several limitations that should be considered when interpreting its findings. Firstly, although the study was conducted in multiple centers across India, the applicability of the findings may be restricted to certain population characteristics and healthcare practices. Another concern arises from potential bias due to variations in data collection methods across multiple CHN hospitals. In this study, DBS samples were collected 36 hours after birth. However, the optimal timing for blood sample collection for detecting MMA during NBS is preferably after 72 hours of birth (Jin et al., 2022). Additionally, the study focused on short-term responses to interventions, lacking a long-term follow-up to assess sustained effects.Lastly, the lack of ethnic diversity in the study population might influence the prevalence of specific conditions and their interactions with maternal factors.

Nevertheless, this study underscores the fact that the metabolic alterations observed during the NBS are not solely determined by IEMs. Therefore, conducting comprehensive assessments, including those on maternal medical history and dietary information, can help in differentiating between true-positive results related to IEMs and false-positive results influenced by external or maternal factors. The proper interpretation of NBS results ensures timely intervention and management for infants with true metabolic disorders while preventing unnecessary stress for families due to false-positive results.

This study highlights the role of vitamin B12 deficiency in mothers, which influences the metabolic levels of methylmalonic acid in their children, and leads to high false-positive rates of MMA detection during NBS. Administering intramuscular vitamin B12 to the mothers effectively reversed the observed metabolic abnormalities. This study adds to the literature that raises concerns regarding “pseudo-MMAs” detected during NBS. A holistic approach to interpreting NBS results—encompassing not only IEMs in infants but also maternal health and external factors—is needed. Also, the knowledge of this should avoid babies being diagnosed with Infantile Tremor Syndrome in future due to maternal vitamin B12 deficiency affecting babies at a later age – which is still being reported from time to time (Kesavan et al, 2023).