Mutations in the lysosomal enzyme-targeting pathway and persistent stuttering

Abstract

Background: Stuttering is a disorder of unknown cause characterized by repetitions, prolongations, and interruptions in the flow of speech. Genetic factors have been implicated in this disorder, and previous studies of stuttering have identified linkage to markers on chromosome 12.

Methods: We analyzed the chromosome 12q23.3 genomic region in consanguineous Pakistani families, some members of which had nonsyndromic stuttering and in unrelated case and control subjects from Pakistan and North America.

Results: We identified a missense mutation in the N-acetylglucosamine-1-phosphate transferase gene (GNPTAB), which encodes the alpha and beta catalytic subunits of GlcNAc-phosphotransferase (GNPT [EC 2.7.8.15]), that was associated with stuttering in a large, consanguineous Pakistani family. This mutation occurred in the affected members of approximately 10% of Pakistani families studied, but it occurred only once in 192 chromosomes from unaffected, unrelated Pakistani control subjects and was not observed in 552 chromosomes from unaffected, unrelated North American control subjects. This and three other mutations in GNPTAB occurred in unrelated subjects with stuttering but not in control subjects. We also identified three mutations in the GNPTG gene, which encodes the gamma subunit of GNPT, in affected subjects of Asian and European descent but not in control subjects. Furthermore, we identified three mutations in the NAGPA gene, which encodes the so-called uncovering enzyme, in other affected subjects but not in control subjects. These genes encode enzymes that generate the mannose-6-phosphate signal, which directs a diverse group of hydrolases to the lysosome. Deficits in this system are associated with the mucolipidoses, rare lysosomal storage disorders that are most commonly associated with bone, connective tissue, and neurologic symptoms.

Conclusions: Susceptibility to nonsyndromic stuttering is associated with variations in genes governing lysosomal metabolism.

Figure 1. Pedigree and Distribution of GNPTAB Glu1200Lys Mutation in Family PKST72

Subjects 77 and 89 (green ovals) were homozygous for the mutation but were unaffected, suggesting nonpenetrance. Subjects 65, 69, and 83 (pink ovals) were affected but differed from the other affected subjects with respect to the haplotype surrounding the gene for the alpha and beta subunits of N-acetylglucosamine-1-phosphate transferase (GNPTAB). Double lines indicate consanguineous unions. Squares denote male family members, circles female members, solid symbols affected members, and slashes deceased members.

Figure 2. Mutations Found in GNPTAB, GNPTG, and NAGPA and Alignment of Their Amino Acid Sequences in Different Species

Shaded areas indicate amino acid changes resulting from mutations in various species. In NAGPA, p.Phe513SerfsX113 denotes a frame-shift mutation that changes phenylalanine 513 to serine and creates a new reading frame that ends in a stop 113 amino acids downstream (see Fig. 4 in the Supplementary Appendix, available with the full text of this article at NEJM.org). GNPTAB denotes the N-acetylglucosamine-1-phosphate transferase gene encoding the subunits alpha and beta, GNPTG the N-acetylglucosamine-1-phosphate transferase gene encoding the subunit gamma, and NAGPA the N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase gene.

Figure 3. Tagging for Transport of Lysosomal Enzyme

In the first step, GlcNAc-phosphotransferase (the alpha, beta, and gamma subunits of which are encoded by the GNPTAB and GNPTG genes) catalyzes the covalent linkage of GlcNAc-1-phosphate from uridine diphosphate (UDP) to the terminal mannose residues of the N-linked oligosaccharides on enzymes destined for the lysosome. In the second step, NAGPA (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase), also known as uncovering enzyme, removes one GlcNAc group, thereby exposing mannose-6-phosphate (M6P), which acts as the targeting signal. Enzymes with this targeting signal are then routed through the Golgi apparatus to the lysosome.