A novel mitochondrial m.4414T>C MT-TM gene variant causing progressive external ophthalmoplegia and myopathy.

We report a novel mitochondrial m.4414T>C variant in the mt-tRNAMet (MT-TM) gene in an adult patient with chronic progressive external ophthalmoplegia and myopathy whose muscle biopsy revealed focal cytochrome c oxidase (COX)-deficient and ragged red fibres. The m.4414T>C variant occurs at a strongly evolutionary conserved sequence position, disturbing a canonical base pair and disrupting the secondary and tertiary structure of the mt-tRNAMet. Definitive evidence of pathogenicity is provided by clear segregation of m.4414T>C mutant levels with COX deficiency in single muscle fibres. Interestingly, the variant is present in skeletal muscle at relatively low levels (30%) and undetectable in accessible, non-muscle tissues from the patient and her asymptomatic brother, emphasizing the continuing requirement for a diagnostic muscle biopsy as the preferred tissue for mtDNA genetic investigations of mt-tRNA variants leading to mitochondrial myopathy.

A new diagnostic assay for glycogen storage disease type II in mixed leukocytes.

We have established a new method for the enzymatic diagnosis of glycogen storage disease type II (Pompe disease or acid maltase deficiency) using mixed leukocytes. The method employs glycogen and 4-methylumbelliferyl-alpha-D-glucopyranoside (4MU-alphaGlc) as substrates for measuring the lysosomal acid alpha-glucosidase (acid alphaGlu) activity, and incorporates acarbose to eliminate the interference of unrelated alpha-glucosidases (predominantly maltase-glucoamylase). It is shown that 3.0 micromol/L acarbose completely inhibits the maltase-glucoamylase activity at pH 4.0, but the lysosomal acid alphaGlu activity by less than 5%. With this method, we determined the acid alphaGlu activity in mixed leukocytes from 25 patients with glycogen storage disease type II (2 infantile and 23 late-onset cases), one GAA2/GAA2 homozygote and 30 healthy subjects. In the assay with glycogen as substrate, the addition of acarbose created a clear separation between the patient and the control ranges. In the assay with 4MU-alphaGlc as substrate, the two ranges were fully separated but remained very close despite the use of acarbose. The separation of the patient and normal ranges was improved considerably by taking the ratio of acarbose-inhibited over uninhibited activity. A GAA2/GAA2 homozygote was correctly diagnosed with 4MU-alphaGlc but misdiagnosed as patient when glycogen was used as substrate. We conclude that the inclusion of 3.0 micromol/L acarbose in the assays with glycogen and 4MU-alphaGlc substrates at pH 4.0 allows for the specific measurement of lysosomal acid alphaGlu activity in mixed leukocytes, thus enabling a reliable diagnosis of glycogen storage disease type II in this specimen.