Letter to the Editor

DNA-based diagnosis of mucolipidosis type IIIA and mucopolysacchariodisis type VI in a Chinese family: A chance of 1 in 7.6 trillion

Mucolipidosis II and III alpha/beta (ML II/III alpha/beta) are rare autosomal recessive lysosomal storage diseases that are caused by a deficiency of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the enzyme responsible for the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. A Brazilian patient suspected of having a very mild ML III was investigated using whole next-generation sequencing (NGS). Two mutations in the GNPTAB gene were detected and confirmed to be in trans status by parental analysis: c.1208T>C (p.Ile403Thr), previously reported as being pathogenic, and the novel mutation c.1723G>A (p.Gly575Arg). This study demonstrates the effectiveness of using whole NGS for the molecular diagnosis of very mild ML III alpha/beta patients.

UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and β subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αβ. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αβ patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and β subunits.

Background: Mutations in GNPTAB cause the lysosomal disorders mucolipidosis II and III αβ.

Results: All reported missense mutations were studied and showed various consequences on its gene product, αβ GlcNAc-1-phosphotransferase.

Conclusion: Domains responsible for catalytic activity and lysosomal hydrolase recognition were identified.

Significance: Analysis of patient mutations provided new insight into the functional domains of αβ GlcNAc-1-phosphotransferase.

Mucopolysaccharidoses (MPS) are caused by the deficiency in the metabolism of one or more types of mucopolysaccharides or glycosaminoglycans (GAGs). Mucolipidoses (ML) are a group of genetic disorders in which both glycosaminoglycans (GAGs) and sphingolipids build up in the body. Both of MPS and ML belong to lysosomal storage diseases and show similar clinical manifestations. Distinction of these two types of diseases has not been always possible using conventional clinical diagnoses. Genetic test provides a definitive diagnosis for ML and MPS diseases.

The initial clinical diagnosis had suspected the proband as either MPS or ML. To verify the clinical diagnosis, linkage analysis was performed with a panel of microsatellite markers flanking 10 candidate genetic loci for mucopolysaccharidosis and 2 loci for mucolipidosis. Two-point logarithm of odds (lod) scores was calculated using Linkage Package 5.2 program. Direct DNA sequence analyses of GNPTAB in the family members were performed.

By using linkage and mutational analyses, we have identified that the family members contain compound heterozygous mutations of p.R364X and c.2715 + 1 G > A in the GNPTAB gene. We determine the family as MLIII based on the DNA-test and clinical diagnoses.

Our study confirms the pathological relationship between the patients' genotype and phenotype in the clinical ML manifestation, and suggests that DNA-based diagnosis serves as a better way to define ML and MPS.

Mucolipidosis type II (ML II) is a fatal, autosomal recessive, lysosomal storage disorder characterized by severe clinical and radiologic features. ML II results from mutations in alpha and beta subunits, encoded by the GlcNAc-1-phosphotransferase gene (GNPTAB). Most of the 40 different GNPTAB mutations reported so far are insertions and deletions predicting diverse types of aberrant proteins. Alu mobile elements have however never been involved in these events up to now. The Italian ML II patient of our study showed an Alu retrotrasposition in GNPTAB exon 5. The Alu insertion mutation (NM_024312.3:c.555_556insHSU14569) generated a transcript with a skipping of the target exon 5 and a frameshift p.S122fs, causing a premature translation termination codon at position 123. This insertion mutation was found in compound heterozygosity with the frameshift p.S887KfsX33, resulting from a new mono-nucleotide duplication (c.2659dupA) that occurred in GNPTAB exon 13. A possible involvement of cis-splicing elements having an exonic location, such as exon enhancers (ESEs), is discussed as mechanism that led to the production of the aberrant mRNA splicing.