Multiple serum protein abnormalities in carbohydrate-deficient glycoprotein syndrome: pathognomonic finding of two-dimensional electrophoresis?

To the Editor: Labor hereditary optic neuropathy is a maternally inherited disease associated with mutations of mitochondrial DNA (1,2). A G to A substitution at position 11778 converts a highly conserved arginine to histidune in the fourth subunit of NADH dehydrogenase (EC 1.6.99.3). This mutation causes the loss of an SfaNI restriction site (1) and creates a new Mae III restriction site (3). We developed a rapid detection of this mutation by a simplification of DNA extraction followed by polymersee chain reaction (PCR) amplification (4) and digestion with restriction enzymes, SfaNI or Mae Ill. Frozen EDTA-anticoagulated blood samples (20 1tL) were treated three times with 1 mL of a solution of 10 mmol of TrisHCl and 1 mmol of EDTA per liter (pH 8.0) at 4#{176}C. After centrifugation at 11 000 x g for 2 miii, the pellet was frozen at -70#{176}C for 5 mm, then heated at 95#{176}C for 5 mm, after which PCR amplification was performed without delay. Primer sequences used for this purpose were 11618-11637 (light strand; 5’-GCATACTCTFCAATCAGCCA-3’) and 11893-11874 (heavy strand; 5’rFCTCCCAGTAGGTTAATAG-3’). The following solution was prepared: per liter, 200 pmol of each deoxynucleotide triphosphate (dATP, dGTP, CICTP, and dTFP), 0.10 tmol of each oligonucleotide primer, 50 mmol of potassium chloride, 1.5 mmol of magnesium chloride, 0.1 g of gelatin, and 10 mmol of Tris HC1 (pH 8.3). We added 50 L of this solution and 1 U ofTaq DNA Polymerase (from Perkin Elmer Cetus, Norwalk, CT, or Boehringer) to the blood pellet and subjected the mixture to 25 cycles of PCR in a DNA thermal cycler (PTC-100; MJ Research Inc., Watertown, MA), with each cycle consisting of denaturation at 94#{176}C for 1 mm, annealing at 52#{176}C for 1 mm, and extension at 72#{176}C for 2 mm. For FIg.1. Polyacrylamldo gel electrophoresisof restrictiondigests with enzymes SfaNl or Mae Illfrom a normal individual (N) andfrom two patients with Leber disease, one being homoplasmic (L) and the other one heteroplasmlc (Ui) C Isa controlcorrespondingto undigested amplified DNA