Abstract
Monoamine oxidase A (MAO‐A) deficiency is a rare inborn error of metabolism with impaired degradation of biogenic amines including 5‐hydroxytryptamine (5‐HT), resulting in borderline intellectual disability and behavioral abnormalities. Genetic variants in MAOA need functional confirmation to enable a definite diagnosis. To this end, we developed an inexpensive, simple and nonradioactive MAO‐A activity assay based on the conversion of 5‐HT into 5‐hydroxyindoleacetic acid (5‐HIAA). Fibroblast cell lysates were incubated with 5‐HT and aldehyde dehydrogenase to allow 5‐HIAA production. 5‐HIAA was quantified using high‐performance liquid chromatography with fluorimetric detection. We optimized reaction mixture components, pH, and substrate concentration and tested linearity and specificity of the assay. We verified the functional validity of the enzyme assay using fibroblasts of controls, female mutation carriers and MAO‐A deficient patients. This included a newly described patient with a novel MAOA variant (c.1336G>A, p.(Glu446Lys)), who represents the fifth MAO‐A deficiency family so far. The optimized enzyme assay showed good linearity and specificity. Application to clinical samples showed a 100% differentiation of affected patients (with negligible MAO‐A enzyme activity) and controls or mutation carriers. In conclusion, the described MAO‐A activity assay is easy to implement and can readily be used to test the pathogenicity of variants in the MAOA gene in a clinical setting. Especially in this era of whole‐exome (and whole‐genome) sequencing, this functional assay fulfills a clinical need for functional confirmation of a suspected diagnosis of MAO‐A deficiency.