Introduction 

Like many loss-of-function diseases, methylmalonic acidemia (MMA) is extremely rare with an incidence rate of 1 in 50,000.¹ However, its rarity hasn’t prevented extensive research from being poured into the causes, diagnosis, and treatment of the disease. This article will provide an insight into the variations and causes of MMA will be provided as well as a discussion of the varying diagnostic tools used for detection. 

Cause 

As a general rule, any disease with the name acidemia refers to there being toxic amounts of excess acid in the body. In MMA, the toxic levels of excess methylmalonic acid are due to an interruption in the metabolic pathway that’s in charge of breaking down methylmalonyl-CoA to succinyl-CoA.² This interruption has two potential culprits; either the methylmalonyl-CoA mutase (MCM) enzyme or cobalamin which is also known as vitamin B12.² Normally, MCM and vitamin B12 work together to break down methylmalonyl-CoA, but when either are made defective by a mutation, they cannot break down methylmalonyl-CoA which leads to excess methylmalonic acid accumulation and MMA.³ 

When diagnosing MMA, one of the key differences to distinguish is whether the problem lies with the MCM enzyme or with vitamin B12 (cobalamin). In other words, distinguishes whether the MMA is a result of a cobalamin disorder or a deficiency of the MCM enzyme. Although determining the cause of MMA in such detail may seem redundant, it’s essential to recognise what the most effective treatment will be. 

Symptoms  

The severity of MMA symptoms can vary depending on which specific gene is mutated. Often symptoms include:

• Vomiting
• Dehydration
• Hepatomegaly (an enlarged liver) 
• Hypotonia (reduced muscle tone)
• Tiredness
• Neurological manifestations (ie seizures or strokes)⁴

The non-specific nature of these symptoms means MMA is often misdiagnosed. 

Diagnosis 

As the list of symptoms suggests, it is hard to diagnose MMA not only due to its rarity but also the fact that many of its symptoms are broad and applicable to various health conditions. When multiple conditions share the same symptoms a differential diagnosis is hypothesized, this describes a list of the potential causes for a patient’s symptoms.⁵ A working hypothesis is then formed as testing occurs to eliminate conditions and nominate the most likely condition. Despite this, the effects of MMA appear in early infancy, so diagnosis of MMA often occurs before the age of 12 months. If a metabolic disorder such as MMA is suspected, often the first step is a urine and or blood test.⁵ 

As many parents will be aware, new-born babies in the UK are recommended a blood test where a few drops of blood are taken by pricking the baby’s heel to be screened for rare and serious conditions such as cystic fibrosis and inherited metabolic diseases like phenylketonuria.⁶ MMA is part of newborn screening in countries like Italy, Spain and the USA but not in the UK.¹

Blood Testing 

After blood has been collected it is screened for threshold levels of certain markers that can indicate MMA in support of the symptoms. These markers include:

• Low bicarbonate levels 
• Low blood glucose levels
• High blood ammonia levels 
• High levels of methylmalonic acid¹

However, even if these markers are met by the results of the blood test, more analysis needs to be done to make the transition from a presumptive or working diagnosis to a definitive diagnosis. Another similar disorder that has the same blood test results as MMA but requires a different treatment plan is Propionic Acidemia (PA).²

PA is caused by a deficiency in the propionyl-CoA carboxylase (PCC) enzyme which is upstream of the MCM pathway. This disorder is rarer than MMA with an incidence rate of 1 in 100,000 to 150,000.¹ 

Urine Testing 

Alongside blood testing, urine tests are often performed. When it comes to metabolic diseases, urine tests can provide a wealth of information because metabolic waste is eliminated in the urine. By analyzing levels of metabolic waste one can paint a picture of which metabolic pathways might be defective or abnormal. Ultimately, analysis of the organic acids in the urine is the most effective way to distinguish between PA and MMA. 

Urine samples are recommended to be taken on an empty stomach where the metabolic waste is unaltered by any recent food intake. Samples are then analyzed by gas chromatography/mass spectrometry (GC/MS) where the liquid sample is vaporized and separated into its components by ionization and fragmentation.² If the results show high levels of methylmalonic acid, the patient can be definitively diagnosed with MMA.² On the other hand, if the results show high levels of propionic acid and propionyl glycine, the patient is definitively diagnosed with PA instead.² 

As alluded to earlier, to decide what the most effective form of treatment will be, one needs to know whether the MMA will be vitamin B12 responsive, i.e. whether cobalamin or MCM is defective and therefore causing MMA. A urine test can differentiate these subtypes of MMA by detecting the methylmalonic acid concentration. If the urine sample contains less than 1000 mmol of methylmalonic acid per mol creatinine the defect is likely with cobalamin synthesis or function.² Alternately, if the urine sample contains more than 1000 mmol of methylmalonic acid per mol creatinine the defect will be with MCM synthesis or function.² 

From here, most cobalamin defective forms of MMA are responsive to vitamin B12 replacement therapies. However, MCM subtypes need further characterisation. 

Enzyme Activity Assay 

To delve further into the subtypes of MCM defective MMA, the mutation to the MCM enzyme can either cause complete deficiency (mut0) or partial deficiency (mut-), where the MCM enzyme is either completely inactive or partially inactive respectively.⁷ Both subtypes tend to be unresponsive to vitamin B12 therapy, so other treatments are needed. To determine the level of MCM deficiency that’s causing MMA (mut0 or mut-), enzyme activity assays are performed.

Fibroblasts describe a cell type most common in connective tissue. They’re often used for enzyme activity assays because they’re easy to culture. There are many different laboratory techniques used to do an enzyme activity assay including thin layer chromatography, electrophoresis, high-performance liquid chromatography (HPLC), paper chromatography and more, yet the pros and cons of each deem HPLC the more popular method.² 

To measure how well an enzyme is functioning by HPLC, one measures the levels of reactant and product of the target enzyme. For example, if the enzyme is completely inactive, there would be no product and excess reactant detected by HPLC, and if the enzyme is partially inactive, there would be some product being made but not to normal levels and still an excess of reactant. HPLC exploits the different chemical properties of the product and reactant to identify what’s what. When testing the MCM enzyme, the reactant is methylmalonyl-CoA and the product is succinyl-CoA. Low levels of succinyl-CoA indicate a mut- mutation to MCM, and even lower levels indicate a mut0 mutation to MCM. 

Treatments 

The potential severity of MMA means that often initial treatment occurs while the working hypothesis is being investigated so that no time is wasted.⁵ The main short-term treatment that applies to all acidemia disorders is a protein-restricted diet.¹ Protein is the largest source of the key building blocks that enter many metabolic pathways, by limiting the number of those building blocks in the body, the problematic metabolic pathway slows down and limits the toxic build-up as a result. It’s basically removing the fuel from the fire.

After a definitive diagnosis of the MMA as well as the subtype of the disorder has been concluded, a long-term treatment plan can be made. For cobalamin deficient MMA, the main treatment is vitamin B12 supplementation.¹ By replacing the deficient levels of cobalamin, the normal metabolic pathway can be restored.

Previously, medical foods with specific amino acid modifications have been used as treatment, again they work using the ‘less fuel for the fire’ principle.⁸ Although these diets work to treat MMA, as a long-term treatment they can lead to other complications down the line. Alternatively, liver transplants have been implemented in more extreme cases of MMA.⁸ 

Summary 

Ultimately, MMA is a more complex disorder than it first appears with its different causes and subtypes as well as its nonspecific symptoms which make it tricky to diagnose quickly. Diagnosing any disease starts with a good knowledge of the root cause, which in this case means doing a few diagnostic tests like blood and urine tests and enzyme activity analysis.