Helena Ribeiro

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

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 (α, β and γ) 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 α/β and the γ subunits.

Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations

SummaryMucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a defect in the iduronate-2-sulfatase gene (IDS). Alternative splicing of the IDS gene can occur and the underlying regulatory mechanism may be rather complex. Nevertheless, little information is available on the role of variations at the IDS locus in the splicing process. Here we report that splice mutations at the IDS locus are an important source of MPS II pathogenicity, accounting for almost 56% of Portuguese cases. Among 16 unrelated Portuguese MPS II patients, 15 different mutations were identified: six intronic splice mutations (c.104−2AG, c.241−2A>G, c.241−1G>A, c.418+1G>A, c.880−8AG and c.1181−1G>C); two exonic splice mutations (c.1006G>lC and c.1122C>T); five missense mutations (D269V, D69V, D148N, R88C and P86L); one nonsense mutation (Q465Ter); one total IDS gene deletion; and one rearrangement involving a IDS gene inversion. Furthermore, nine of the 15 detected mutations affected the usual splicing pattern at the locus. Some of them are responsible for dramatic changes in the splicing mechanism. For example, the substitution mutation, c.418+1G>A, revealed the presence of an exonic sequence inside intron 3. Our study provides evidence that the IDS locus is prone to splicing mutations and that such susceptibility is particularly high in exon 3 and neighbouring regions. Consequently, mutation screening of the IDS gene cannot be restricted to gDNA examination. Unless cDNA analysis is also conducted, misclassifications as silent or missense mutations can be produced and even uncharacteristic splice-site mutations can be misinterpreted as classic splicing defects that may generate severe, unconventional splicing alterations.

Lysosomal multienzymatic complex-related diseases: a genetic study among Portuguese patients

Coutinho MF, Lacerda L, Macedo‐Ribeiro S, Baptista E, Ribeiro H, Prata MJ, Alves S. Lysosomal multienzymatic complex‐related diseases: a genetic study among Portuguese patients.

Molecular characterization of Portuguese patients with mucopolysaccharidosis IIIC: two novel mutations in the HGSNAT gene.

To the Editor: Mucopolysaccharidosis IIIC (MPS IIIC, Sanfilippo syndrome C) belongs to a class of lysosomal storage disorders known as mucopolysaccharidosis characterized by a deficiency in one of a group of enzymes responsible for the catabolism of glycosaminoglycans (1). MPS IIIC is caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: a-glucosaminide Nacetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate (1). Hallmark symptoms of MPS IIIC include mental retardation and hearing loss as well as relatively minor visceral manifestations (1, 2). It is known that the gene encoding N-acetyltransferase – HGSNAT – is located on chromosome 8p11.1andcontains18exons (3, 4).ThecomplementaryDNA(cDNA)codes foraproductof 635amino acids (previously named transmembrane protein 76 – TMEM 76), of which the N-terminal 30 amino acids are predicted to form a cleavable signal peptide, while along the remainder of the protein, there are11 transmembranedomainsandupto5N-linked glycosylation sites (3, 4). However, because these findings only came to light in 2006 when the HGSNAT gene was identified by two independent groups (3, 4), the molecular defects underlying MPS IIIC still remain largely uncharacterized. We have developed a new molecular method to characterize patients with MPS IIIC for the HGSNAT gene through cDNA analysis and identified two novel mutations: an insertion (c.525dupT) and a splice-site mutation (c.3722A/G), both of which are very likely deleterious to the function of HGSNAT. Our sample included three unrelated Portuguese patients with MPS IIIC whose clinical diagnosis was biochemically confirmed by demonstrating abnormal excretion of heparan sulfate in urine and HGSNAT deficiency in fibroblasts. Total cellular RNA was isolated from cultured fibroblasts using the High Pure RNA Isolation Kit’ (Roche, Basel, Switzerland) and reverse transcribed using the First-Strand cDNA Synthesis Kit’ (Amersham Biosciences, Munich, Germany). We designed specific primers to amplify the HGSNAT cDNA in seven overlapping fragments. Genomic DNA was also isolated from cultured fibroblasts, and polymerase chain reaction (PCR) amplification of HGSNAT exons including adjacent intronic regions was performed with specific primers. Primers and PCR conditions are provided as online supplementary material. The three patients only harbored two different mutations c.525dupT and c.372-2A/G (Table 1), both of which were previously unreported. Their presence was confirmed in patient’s cDNA and genomic DNA, and none was detected in 100 chromosomes from healthy Portuguese. Both mutations are very likely deleterious to the function of HGSNAT because they predictably result in a total loss of HGSNAT protein function. The insertion mutation c.525dupT causes the introduction of a premature STOP codon downstream that results in the translation of a product with 445 amino acids less than the normal protein. The splice-site mutation c.3722A/G leads to the skipping of exon 4 that also causes the introduction of a premature STOP codon downstream. Both transcripts will probably be degraded through the cellular mechanism of nonsensemediated messenger RNA (mRNA) decay, which is well known to be responsible for the elimination of mRNAs that contain premature termination codons (5). In full agreement with the finding of Hřebı́ček et al., when HGSNAT was sequenced, all studied individuals revealed the presence of an alternative transcript unlikely to be functional because it presented exons 9 and 10 spliced out (4). However, it can be excluded as causative of the MPS IIIC phenotype because of being a minoritary transcript that also appeared in an unaffected individual.

Molecular and computational analyses of genes involved in mannose 6-phosphate independent trafficking

The newly‐synthesized lysosomal enzymes travel to the trans‐Golgi network (TGN) and are then driven to the acidic organelle. While the best‐known pathway for TGN‐to‐endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP‐2, implicated in the delivery of β‐glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P‐independent receptors, we screened the genes that encode for LIMP‐2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease.