Biochemical analysis of GNPTAB missense mutations associated with ML II

Mucolipidosis type II and type III (ML II and III) are rare autosomal recessive disorders of lysosomal hydrolase trafficking respectively caused by completely absent or reduced activity of the enzyme GlcNAc-phosphotransferase, which catalyzes the initial step in the synthesis of mannose-6-phosphate recognition marke. This heterohexameric enzyme composed of three subunits (alpha2 beta2 gamma2), is a product of two distinct genes GNPTAB and GNPTG. Mutations in GNPTAB result ML II and ML III, while mutations in GNPTG are only associated with ML III. To date more than 100 different GNPTAB mutations have been described, causing either ML II alpha\beta or ML III alpha\beta. Although splicing and frameshift mutations are usually associated with more severe phenotypes and missense mutations with milder ones, this typical pattern is not observed for all ML II patients. Here we report the impact of two GNPTAB missense mutations upon the protein: while W81L occurred in the portion of the gene that encodes the apha-subunit, R986C affected a genomic region encoding the beta subunit. To address this issue, the entire coding region of the wild-type GNPTAB was cloned into the pcDNAHisMax TOPO vector and the c.440delC (A147AfsX5), c.2956T>C (R986C) and c.242G>T (W81L) were introduced on this vector using the QuikChange Site-directed Mutagenesis kit. The presence of additional mutations, resulting from possible enzymatic misincorporation, was excluded by sequencing all constructs. COS7 cells were transfected with control and mutant plasmids using Lipofectamine 2000 reagent. Protein expression levels and subcellular location were determined through Western Blot and Immunofluorescence, respectively. Results and Conclusions: We analyzed the protein expression levels of three GNPTAB mutations: c.242G>T (W81L), c.2956T>C (R986C) andc.440delC (A147AfsX5). The frameshift c.440delC (A147AfsX5), predicting to introduce a premature stop codon, was used as negative control. and, as expected, no GNPTAB protein product was detectable. Instead, the analysis of both missense mutations, c.2956T>C (R986C) and c.242G>T (W81L), revealed a decrease in GNPTAB protein expression, compared to the control wild type. This concurs with a previous computational assessment by the Polyphen and SIFT algorithms, predicting that the 2 mutations were likely to be potentially damaging. In addition, computational analysis (http://www.ensembl.org/) revealed that both missense mutations occurred at evolutionarily conserved amino acid residues. The results of all these approaches correlate with the severe ML II phenotype of the patients

Molecular characterization of the Portuguese patients with defects in GlcNAc-phosphotransferase: a key enzyme in the M6-P dependent lysosomal trafficking

Introduction: GlcNAc-phosphotransferase is one of the enzymes responsible for the formation of M6P residues and plays a key role in lysosomal trafficking, since most soluble acid hydrolases reach these organelles through the M6P pathway. It is composed of six subunits (α2β2γ2), products of two genes recently cloned: GNPTAB (mutated in mucolipidosis II/IIIA patients) and GNPTG (mutated in MLIIIC patients). Methods: Using both gDNA and cDNA extracted from patient’s fibroblasts, we performed a molecular study of both genes in 13 MLII/III patients (10Portuguese, 1Finnish, 1Spanish of Arab origin and 1Indian). Expression studies were performed by quantitative real-time PCR. Results: We identified 11 different mutations, 8 of them novel: 6 in the GNPTAB gene (c.121delG;c.440delC;c.2249_50insA;W81L;I403T and E667) and 2 in the GNPTG gene (c.610-1G.T and c.639delT). Interestingly, although the MLII-causing mutations have been mostly found to be private or rare, there is one (c.3503_3504delTC) that shows a broad distribution having been detected among different populations. This same mutation was also the most frequent one in our patients. Such distribution pattern prompted us to perform a haplotypic study. We analysed 37 patients (23Italians, 8Arab Muslims, 1Turkish and 5Portuguese) for 3 intragenic polymorphisms and 2 microsatellite markers flanking the GNPTAB gene, identifying a common haplotype. Regarding the mRNA expression studies, real-time results suggest the existence of feedback regulation mechanisms between α/β and the γ subunits. Discussion/Conclusion: This work enabled the establishment of a strong genotype-phenotype correlation, which is of crucial importance to an improved genetic counselling for ML families. The sharing of an ancestral haplotype by patients carrying the deletion implies a common origin of this mutation, while the higher level of diversity observed at the most distant locus indicates that it is a relatively ancient one. The developed strategies constitute valuable tools that allow carrier detection and prenatal- molecular diagnostics of these diseases