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A role for inherited metabolic deficits in persistent developmental stuttering

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Abstract

Stuttering is a common but poorly understood speech disorder. Consistent evidence for the involvement of genetic factors in stuttering has motivated studies aimed at identifying causative genetic variants that could shed light on the underlying molecular and cellular deficits in this disorder. Such studies have begun to identify causative genes. The purpose of this review is to summarize the gene discoveries to date, and to cover the subsequent functional studies that are beginning to provide insights into how these gene mutations might cause stuttering. Surprisingly, the first variant genes to be associated with stuttering are those encoding the lysosomal targeting system, GNPTAB, GNPTG, and NAGPA. Although mutations in NAGPA have not been associated with a disorder in humans, mutations in GNPTAB and GNPTG cause mucolipidosis types II and III, which are rare autosomal recessive lysosomal storage disorders, associated with pathology of bone, connective tissue, liver, spleen, and brain. Analysis of mutations in these genes has so far identified predominantly missense mutations in stuttering, in contrast to the truncating and other mutations that result in very low GNPTAB/G enzyme activity and are historically associated with mucolipidosis. Genetic evidence for the role of lysosomal targeting mutations in stuttering has now been buttressed by biochemical studies of the mutant enzymes found in this disorder. While data on the GlcNAc-phosphotransferase encoded by GNPTAB/G remains limited and only suggestive, a study of the enzyme encoded by NAGPA has shown that the mutations found in stuttering reduce the overall cellular activity of this enzyme by about half, and that they result in deficits in intracellular processing and trafficking that lead to a reduced cellular half life. How these deficits result in the presumed speech-specific neuropathology associated with stuttering is not yet known. However these findings have opened several new lines of inquiry, including studies in mice carrying human stuttering mutations, that represent promising approaches to this disorder.

Highlights

► Linkage studies are rapidly identifying new loci for stuttering. ► To date mutations in GNPTAB, GNPTG, and NAGPA have been associated with stuttering. ► These genes encode the lysosomal enzyme targeting pathway, defective in mucolipidosis. ► Stuttering individuals studied to date showed no symptoms of mucolipidosis. ► Deficits in intracellular trafficking and reduced enzyme half-life are present.

Introduction

Stuttering is a disorder affecting the flow of speech, characterized by repetitions or prolongations of syllables and words, and by interruptions in the flow of speech known as blocks. Stuttering typically arises in young children ages three to five, where it affects up to five percent of children in this age group. The majority of affected children (75–80%) recover, either spontaneously or with the aid of speech therapy. Nevertheless, in some the disorder persists, leading to a frequency of stuttering in the general population of approximately one percent. Our studies are focused on persistent developmental stuttering, which occurs in populations and language groups worldwide, and despite being recognized and well described since ancient times, little is known about its causes [1]. However substantial evidence has accumulated that genetic factors play an important role in stuttering. Twin studies have repeatedly demonstrated a substantial genetic component to this disorder, with heritability estimates ranging as high as 0.8 [2], [3], [4], [5], [6], [7]. Adoption studies have also been performed, and while the statistical power of these studies has been limited, no support has been obtained for a substantial role of environment in this disorder [8], [9]. While stuttering displays clear familial aggregation [10], [11], segregation analyses of the disorder in families have produced mixed results [12], [13], and Mendelian transmission is frequently not observed.

Nevertheless, based on the strong overall evidence for genetic factors in stuttering, a number of genetic linkage studies have been undertaken. The results of these early linkage studies, which analyzed relatively small families from outbred populations, were disappointing. Few convincing linkage scores were obtained, and linkage loci often were not replicated across different studies [14], [15], [16]. This led to an alternative strategy for linkage studies, using large, highly consanguineous families with many affected members. Several such studies have employed consanguineous Pakistani kindreds and have produced strong evidence for linkage. A study of 44 such families identified clear linkage on chromosome 12q that reached genome-wide significance using multiple non-parametric analysis methods [17]. More recently, a study of a single Pakistani family identified linkage on chromosome 3q under a model of autosomal recessive transmission, and generated a maximum LOD score of 4.23 [18]. An additional recent study of a single Pakistani family generated a LOD score of 4.42 on chromosome 16q, again under an autosomal recessive model of inheritance [19]. In both of the latter studies, linkage scores were greatly reduced under other modes of inheritance. Beyond Pakistan, a linkage study has been performed in a large non-consanguineous family from Cameroon that contains many affected individuals. In this family, no single chromosomal locus generated significant linkage scores in the full pedigree. However analysis of different combinations of sub-pedigrees generated significant evidence for linkage on chromosomes 2p, 3p, 3q, and 14 under a recessive mode of inheritance [20]. This suggested that, like other studies in non-consanguineous families, no single highly penetrant gene variant was responsible for the many cases of stuttering observed in this family. Rather gene variants at multiple loci, perhaps brought together by assortative mating, seem to be causative in this family. Taken together, the recent linkage studies of stuttering indicate that highly significant linkage scores can be obtained, that evidence supporting autosomal recessive transmission is frequently observed, and that substantial locus heterogeneity exists for this disorder.

One linkage locus [17] has led to the identification of a causative gene for stuttering. An analysis of the genomic region identified on chromosome 12q initially identified a mis-sense mutation in the GNPTAB gene in 28 affected members of one large Pakistani stuttering family. This mutation substituted a lysine residue for the highly conserved glutamic acid found at amino acid position 1200 in the protein product of this gene. The same mutation was also found in the available affected members of three other Pakistani stuttering families (a total of 3 individuals), as well as in nine unrelated stuttering individuals in the Pakistani and Indian populations [21]. A detailed analysis of the chromosomes containing this mutation revealed that it occurs in a conserved haplotype, indicating that this represents a founder mutation, with a likely single origin rather than arising by multiple mutational events at this position in the gene. Analysis of the existing variation in the chromosomal region surrounding this mutation showed that the conserved haplotype was quite short (approximately 7 kb), suggesting that this mutation is relatively old, having originated perhaps 14,000 years ago [22]. A number of other mutations in the GNPTAB gene were subsequently identified in unrelated individuals who stutter (123 Pakistani, 270 North American subjects) but not in normal controls (96 Pakistani, 276 North American subjects), and several of these mutations were observed in multiple unrelated individuals. Therefore there may be a limited spectrum of mutations in this gene, several of which are repeatedly observed, that are associated with stuttering.

The GNPTAB gene encodes the α and β subunits of the enzyme GlcNAc-phosphotransferase (EC 2.7.8.17), which is involved in the generation of the mannose 6-phosphate (Man-6-P) targeting signal that directs a diverse group of acid hydrolases to the lysosome [23]. These lysosomal hydrolases comprise ~ 60 enzymes capable of metabolizing a great variety of biologic materials, breaking down these macromolecules to their component parts that can then be re-used. Newly synthesized acid hydrolases are modified in the endoplasmic reticulum (ER) where they acquire Asn-linked high mannose glycans and undergo folding, followed by transport to the Golgi complex where Man-6-P tags are attached. These tags allow binding to Man-6-P receptors in the late Golgi followed by transport to the lysosomes.

GlcNAc-phosphotransferase contains the product of another gene designated GNPTG, which encodes the γ subunit of the GNPTAB/G enzyme that enhances the generation of the Man-6-P recognition marker on a subset of the acid hydrolases [24]. Thus GNPTG became a biological candidate gene for stuttering. Sequencing of this gene in unrelated stuttering cases and controls identified a number of different mis-sense mutations associated with stuttering, none of which appeared in controls.

The Man-6-P signal is synthesized by a two-step process (Fig. 1). First, GlcNAc-phosphotransferase covalently links GlcNAc-1-P to specific mannose residues of the Asn-linked high mannose glycans present on the acid hydrolases. In the second step, the GlcNAc is removed, leaving the exposed Man-6-P targeting signal. This second step is performed by the enzyme GlcNAc-1-phosphodiester-N-acetylglucosaminidase (NAGPA, EC 3.1.4.45, also known as the uncovering enzyme (UCE)). This suggested that the NAGPA gene might also be a source of mutations that are associated with stuttering. Evaluation of this gene identified a number of mutations in stuttering cases that were not found in controls. Overall, mutations in GNPTAB, GNPTG or NAGPA were found in approximately 9% of unrelated individuals with familial stuttering [21]. This indicated that mutations that affect the lysosomal targeting pathway can account for a small but significant portion of stuttering.

These findings were surprising because mutations in the GNPTAB and GNPTG genes had been previously well known to be the underlying cause of the lysosomal storage disorders know as mucolipidoses types II and III. Mutations in GNPTAB are causative in mucolipidosis types II (MLII, also known as I-cell disease, MIM #252500) and IIIA (MLIIIA, also known as pseudo-Hurler polydystrophy, MIM #252600) [23], [25], while mutations in the GNPTG are associated with mucolipidosis type IIIC (MLIIIC, variant pseudo-Hurler polydystrophy, MIM #252605) [26], [27]. These three diseases are rare autosomal recessive disorders characterized primarily by abnormalities of bone, connective tissue, liver, heart, and brain. MLII is typically diagnosed at birth and has a severe course, leading to death within the first decade of life. MLIIIA and MLIIIC are less serious diseases with a widely varying course, but they are also ultimately fatal, often in young adulthood [28]. Mutations in NAGPA had not been previously associated with any human disorder. This has been puzzling, as mutations in this gene might be expected to produce mucolipidosis. We hypothesize that the most common manifestation of mutations in NAGPA is persistent developmental stuttering.

Our findings immediately raised the question of whether our stuttering subjects with mutations in these three genes displayed symptoms of MLII or MLIII. A detailed evaluation of a limited number of such subjects (two subjects heterozygous for the p.Ala455Ser mutation in GNPTAB, one homozygous for the p.Arg328Cys mutation in NAGPA, and one heterozygous for the p.Phe513SerfsX113 mutation in NAGPA) at the National Institutes of Health Clinical Center failed to reveal any such symptoms, thus the mutations we identified appeared to be associated with persistent developmental stuttering in the absence of any other medical symptoms. This led to the question of why these mutations, some of which occurred in homozygous form in some stuttering individuals, failed to generate frank MLII or MLIII. Although we currently do not have an answer to this question, we have some suggestive observations. First, mutations associated with MLII are frequently truncating mutations that eliminate or drastically reduce the activity of the GNPTAB/G enzyme [23], [25], [26], [29], while with one exception (discussed further below), the mutations associated with stuttering are mis-sense mutations [21]. It is possible that mutations that cause mucolipidosis cause a more severe deficit in enzyme function than those associated with stuttering. Second, MLII and MLIII are highly penetrant autosomal recessive disorders, associated with homozygosity for mutations in GNPTAB or GNPTG. Most of the individuals in our stuttering sample are heterozygous, carrying a single mutant copy. This observation has led to the question of why parents of children with MLII or MLIII, who are obligate carriers of mutations in GNPTAB or GNPTG do not appear to stutter or display other speech pathologies [30]. Again, the answer to this question is unknown. However, it is possible that truncating mutations associated with mucolipidosis may not be transcribed or if they are, the transcripts are subject to nonsense mediated decay [31]. This would effectively eliminate transcripts from the mutant allele, and leave carriers with transcripts only from the normal allele, although perhaps at reduced levels. Genes containing mis-sense mutations, on the other hand, are likely to be transcribed and translated, and the resulting protein incorporated into the GlcNAC-phosphotransferase enzyme, which exists as an 2α, 2β, 2γ heterohexamer. This could result in the production of normal amounts of an enzyme composed of a mixture of wild type and mis-sense mutant subunits that displays reduced activity.

We mentioned above that all mutations associated with stuttering to date have been mis-sense mutations with one exception. That exception is a 16 bp deletion of NAGPA, resulting in p.Phe513SerfsX113 in the encoded protein. In this mutant protein, the normal phenylalanine at amino acid position 513 is replaced by serine, and the last 2 amino acids and stop codon are missing, leaving an open reading frame that results in the addition of 113 nonsense amino acids until the occurrence of a stop codon. It is of interest that this mutation was found in a 67 year old woman of European descent, who was one of the individuals who underwent a detailed medical examination at the NIH Clinical Center. This individual displayed exceptionally severe lifelong stuttering and no evidence of mucolipidosis.

Section snippets

Biochemical studies of mutant enzymes found in stuttering

While the occurrence of mutations in the GNPTAB, GNPTG, and NAGPA genes made a strong case for the role of these mutations in stuttering, functional evidence for a resulting deficit in the encoded enzymes was initially lacking. Fortunately, because these enzymes had been identified and well studied in the context of the human disease mucolipidosis, a number of important tools for the study of these enzymes, including in vitro and cell-based assays, have been previously developed [32], [33].

Clinical effects of mutations associated with stuttering

While at least some of the mutations associated with stuttering result in enzymes with reduced activity, it is not yet clear how this reduction in enzyme activity leads to stuttering. In general, neurologic deficits are seen in many lysosomal storage disorders. However other very specific pathologies are often observed, where some organs, or cell types within organs, are severely compromised and others largely spared, despite the fact that lysosomal enzymes are expressed widely throughout the

Genetic insights into a broader fraction of stuttering

Mutations in the GNPTAB, GNPTG, and NAGPA genes have been found in less than 10% of unrelated stutterers who have a family history of the disorder [21]. However, the recent linkage studies discussed in Section 1.1 above have given rise to optimism that additional genes, which could explain a larger fraction of stuttering, can be identified [18], [19]. At the present time the causative genes that reside at these loci remain unidentified. It is not possible to know if mutations in these genes

Acknowledgments

We thank Stuart Kornfeld, Thomas Friedman, and Allen Braun for valuable comments on the manuscript. This work was supported by the NIH/NIDCD intramural grant Z01-000046-12 and by the Stuttering Foundation of America.

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    Present address: School of Biological Sciences and Chemistry, Sungshin Women's University, Seoul, Korea 142–732.

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