Results: All subjects of group A, 5 of group B, and 1 of group C, were diagnosed with MSUD, respectively. Among the 11 subjects with normal results, marked elevation of blood leucine levels was observed in 2 sibling cases repetitively, suggesting thiamin-responsive MSUD; genetic analysis is under way. Discussion: Our simple enzymatic assay is a quite useful tool for clinical practice of the disease. It can be a laborious task to detect gene mutations for MSUD because branched-chain α-ketoacid dehydrogenase complex consists of many subunits. Enzymatic diagnosis enables us to focus on cases where genetic analysis is strongly required. We present the first case of a MSUD patient with a mild variant phenotype and bearing a null mutation in the PPM1K gene, which encodes for the regulatory enzyme, protein phosphatase 2Cm, of the branched-chain alpha keto acid-dehydrogenase complex. After completing the direct sequencing of the BCKDHA-E1alpha, BCKDHB-E1beta, DBT-E2 and DLD-E3 genes and considering that genomic rearrangements are the second most common cause of monogenic disorders, patient's DNA was analyzed using the single-nucleotide polymorphism genotype array technology. Results showed a copy neutral homozygous pattern for chromosome 4 compatible with a uniparental isodisomy (UPD) involving the entire chromosome. In other autosomal recessive disorders, homozygosity mapping has provided a hint about the chromosomal location of the probable disease-causing gene. Therefore, as the PPM1K gene is located in region 4q22.1 of chromosome 4, we sequenced the complete coding region of PPM1K (NM_ 152542.3) identifying a homozygous nucleotide change c. 417_418delTA (p.His139fs) in cDNA and genomic DNA from patient. The mutation was only inherited from the father, confirming a paternal UPD. Inmunoblotting assay of the expressed mutant PPM1K showed absence of PP2Cm protein. Our data are consistent with the previously assumed relationship between the loss of PP2Cm activity and an intermediate presentation of MSUD.