Structure of the GM2A gene: Identification of an exon 2 nonsense mutation and a naturally occurring transcript with an in-frame deletion of exon 2

Deficiency of the GM2 activator protein, encoded by GM2A, results in the ra re AB-variant form of GM2 gangliosidosis. Four mutations have been identifi ed, but the human gene structure has been only partially characterized. We report a new patient from a Laotian deme whose cells are deficient in both GM2-activator mRNA and protein. However, reverse transcription (RT)-PCR det ected some normal-sized cDNA and a smaller cDNA species, which was not seen in the RT-PCR products from normal controls. Sequencing revealed that, alt hough the patient's normal-sized cDNA contained a single nonsense mutation in exon 2, his smaller cDNA was the result of an in-frame deletion of exon 2. Long PCR was used to amplify introns 1 and 2 from patient and normal gen omic DNA, and no differences in size, in 5' and 3' end sequences, or in res triction-mapping patterns were observed. From these data we developed a set of four PCR primers that can be used to identify GM2A mutations. We use th is procedure to demonstrate that the patient is likely homozygous for the n onsense mutation. Other reports have associated nonsense mutations with dra matically reduced levels of mRNA and with an increased level of skipping of the exon containing the mutation, thus reestablishing an open reading fram e. However, a recent article has concluded that, in some cases, the latter observation is caused by an artifact of RT-PCR. In support of this conclusi on, we demonstrate that, if the competing, normal-sized cDNA is removed fro m the initial RT-PCR products, from both patient and normal cells, by an ex on 2-specific restriction digest; a second round of PCR produces similar am ounts of exon 2-deleted cDNA.