Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III – identification of eight novel mutations

Abstract

Mucolipidosis II (ML II) and mucolipidosis III (ML III) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. GlcNAc-phosphotransferase is a multimeric transmembrane enzyme composed of three subunits (alpha, beta and gamma) encoded by two genes -GNPTAB and GNPTG. Defects in GNPTAB result in ML II and III whereas mutations in GNPTG were only found in ML III patients. We have performed a molecular analysis of the GNPTAB and GNPTG genes in 13 mucolipidosis II and III patients (10 Portuguese, one Finnish, one Spanish of Arab origin and one Indian). Mutations were identified by the study of both cDNA and gDNA. The GNPTAB and GNPTG mRNA expressions were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The study led to the identification of 11 different mutations. Eight of these mutations are novel, six in the GNPTAB gene [c.121delG (V41FfsX42), c.440delC (A147AfsX5), c.2249_50insA (N750KfsX8), c.242G>T (W81L), c.1208T>C (I403T) and c.1999G>T (p.E667X)] and two in the GNPTG gene [c.610-1G>T and c.639delT (F213LfsX7)]. With regard to the mRNA expression studies, the values obtained by qRT-PCR indicate the possible existence of feedback regulation mechanisms between alpha/beta and the gamma subunits.

Solving a case of allelic dropout in the GNPTAB gene: implications in the molecular diagnosis of mucolipidosis type III alpha/beta

“…In order to search for the molecular defect(s) underlying the GlcNac-1-phosphotransferase impairment in the patient, cDNA analysis of the GNPTAB transcript was performed and the missense mutation c.1196C>T(p.S399F) [8] was identified in heterozygosity. No other alterations were detected through repeated amplification of the GNPTAB cDNA, even though both the patient clinical presentation and the biochemical results were incoherent with the expectations for a carrier individual.…”

“…GNPTAB and GNPTG genomic and cDNA analyses were performed with specific primers according to previously reported conditions [8].…”

Alu-Alu Recombination Underlying the First Large Genomic Deletion in GlcNAc-Phosphotransferase Alpha/Beta (GNPTAB) Gene in a MLII Alpha/Beta Patient

“…We can hypothesise that at least some ML II patients from whom no RNA samples were available and, in whom only one mutant allele has previously been found (Encarnação et al 2009;Tappino et al 2009;Otomo et al 2009;Zarghooni and Dittakavi 2009), might be compound heterozygous for large deletions caused by Alu-mediated mechanisms. The confirmation of that status would be of obvious implications in genetic counselling and prenatal diagnosis, but in which regards to the partially characterised patients from our series (Encarnação et al 2009), RNA samples were available for testing and no gross deletions were found in either of them. Another important point that deserves attention is the fact that patients who have already been classified as homozygous for other GNPTAB mutations may be misclassified if that mutation happens to occur in a region where one of the alleles carries a gross deletion.…”

“…The molecular examination of a ML II patient was initially assessed through routine procedures, which involved amplification and direct sequencing of GNPTAB exons and flanking intronic regions (Encarnação et al 2009). However, the failure to amplify the fragment correspondent to exon 19 and its intronic boundaries from the patient's DNA in face of normal amplification from control DNA, led us to suspect that a large deletion could underlie the molecular defect.…”

“…cDNA Analysis GNPTAB cDNA analysis was performed with specific primers (see Table 1) according to the previously reported conditions (Encarnação et al 2009). …”