Novel method for picking up large heterozygous deletions with semiquantitative PCR in patients with mucolipidosis III alpha/beta

Abstract

Mucolipidosis II (ML II alpha/beta) or I-cell disease is a rare genetic disease in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes: GNPTAB and GNPTG. Although a wide spectrum of mutations in GNPTAB has recently been reported to cause ML II alpha/beta, there are some patients that were previously reported as possessing only one heterozygous mutation while the second one was missing. Here we support the idea that some of these cases may be attributable to the methodological problem of direct sequencing since large heterozygous deletions are undetectable through this method. Methods: We developed a semiquantitative approach by multiplex PCR and capillary electrophoresis. In order to do so, we co-amplified two exon fragments in each PCR reaction, including the one corresponding to GNPTAB exons and an internal fragment that would work as a control (primer pairs for LIPA exons, the gene that codes for acid lipase, another lysosomal enzyme). Results: After testing this technique in the two Portuguese patients that present heterozygous missense mutations but with the second mutation missing, we found evidences that support the idea that, in both patient, the last exons (20 and 21) of the GNPTAB gene are missing in one of the alleles. Conclusion: Here we present the first molecular evidence of the existence of large deletions in the GNPTAB, underlying Mucolipidosis type III as well as a novel method for detecting these types of alterations when present in heterozygous patients. This semiquantitative PCR approach is a valuable tool that allows the screening of large deletions in the GNPTAB gene. In conclusion, ML III cases with only one heterozygous mutation already detected trhpugh direct sequencing methods should be further screened for the possible presence of a large heterozygous deletion mutation