(i) STU 29 family with c.3598G>A variant inGNPTAB gene
The fact that the highest linkage scores were obtained for this variant combined with lack of other plausible genetic variant within the linkage interval suggested increase risk of stuttering when present in either one or two copies 19. Fedyna et al., 27 reported 4/8 unrelated PWS carried atleast one copy of p.Glu1200Lys mutation in GNPTAB gene and established this as a founder mutation in Asian population, originating from Pakistan or India. Recurrence of this lysine variant in heterozygous condition (0.8%) that is segregating with affected status among our south Indian stuttering family favors the founder effect in Asians. Stuttering endophenotypes are homogenous and stable phenotypes. We hypothesized that in a cohort with severe stuttering as an endophenotype, there may be an increased chance to identify lysine variants in homozygous condition. In order to verify this we tested additionally, 26 severe PWS, but identified again only heterozygous lysine variants in three of them; thereby increasing the overall frequency of this variant to 4.4% (4/90*100) with an allele frequency of 2.2% (4/180*100).
High frequency of this variant among south Asian ancestry (2.1%) in the ExAC database, questions its pathogenicity. However, it is highly conserved (Consurf = 8) and a mutation in it disrupts the helical segment that may be crucial for interaction with other subunits or proteins in lysosomal pathway (figure A4).
In a recent animal model study 3- to 8-day old mice pups were engineered to carry two copies of the lysine mutation (Gnptabmut/mut), resulting in significantly longer pauses in their spontaneous vocalizations consistent with some features of human stuttering, but was not found to be lethal as in case of mutations reported in mucolipidosis. This was neither observed in littermates without the mutation (Gnptabwt/wt) nor in heterozygous (Gnptabmut/wt) littermates24. Though the causative role is well established for the homozygous lysine variant in GNPTAB gene, the heterozygous variants were similar to wild-type phenotypically in mouse model. However it is still unclear how a recessive allele in heterozygous condition is causing stuttering phenotype in human model; this trend has been consistently observed not only in our study but in all other reported studies19,21,28. This may be due to (i) a second pathogenic mutation in this gene acting in trans or (ii) other additive genetic factors that may play a causative role or (iii) the gene may have a role in some other unknown pathway. Hence identifying new interacting genes or pathways may clarify the causative role of this gene in stuttering. In fact, dominance and recessiveness are not essentially allelic properties but measured in relation to the effects of other alleles at the same locus. Additionally, dominance may change according to the level of organization of the phenotype and its variations highlight the complexity of understanding genetic influences on phenotypes29.
Two more GNPTAB homozygous mutations p.Ser321Gly and p.Ala455Ser were engineered in mice, that also displayed vocalization deficits traceable to abnormalities in astrocytes of corpus callosum25.