Search for other putative pathways
A segregation based strategy was used to account for those gene variants overlapping with the affected members (276 genes in STU-66 family and 294 in STU-65) but absent in the unaffected members. Seven genesviz., SLC36A1, C1orf167, SYNC, TM4SF1, MUC6, SPTBN5 andMSRB1 were common to both the families with stuttering though they had different sets of multiple variants in each gene.SLC36A1 encodes a proton dependent amino acid transporter, highly expressed in brain. Mutations in this gene involves poor coordination of speech associated with iminoglycinuria and spinocerebellar ataxia 45. Mutations in C1orf167 is associated with neural tube defects.SYNC gene is involved in muscle contraction and is regarded as a marker of neuromuscular disease. TM4SF1 and MUC6 genes form the extracellular components. SPTBN5 gene encodes protein that links membrane lipids, proteins and cytosolic factors to cytoskeletal filaments and also binds to kinesin and actin proteins.MSRB1 gene acts in response to stress and is also involved in actin repolymerization (https://www.genecards.org). Analysis of protein-protein interactions (https://string-db.org/) of the protein products of these seven genes using in silico tool (figure A5), the gene products did not show any interaction with each other but they seem to be involved in a common biological mechanism: within cell or cell-cell communication. These deficits at neuromuscular junctions might contribute to stuttering as speech involves 40,000 neromuscular events per second that act in coordination16.
In each family the downstream analysis of genes that overlap only among affected members resulted in a set of 30/276 genes in STU-66 and 34/294 genes in STU-65. These 64 genes when subjected to pathway browser tool (that interact with Reactome biological pathways), to look for common biological mechanisms, resulted in an enriched gene list of 14 target genes (COL4A2, COL6A3, COL6A6, ITGAX, LAMA5, ADAMTS9, CSGALNACT1, TMOD2 , HTR2B , RSC1A1 , TRPV2, WNK1 , ARSD andSPTBN5) with P-values ranging from 0.125-0.0001 (figure 4). Since the genes that interact significantly tend to have a common function and may be involved in the same pathway, functional dissection of 14 possible candidate genes was attempted to identify the pathways related to stuttering.
Genes like COL4A2, COL6A3, COL6A6 are involved in formation and degradation of collagens17, ITGAX for integrin18 and LAMA5 for laminin19 that together form the extra cellular matrix (ECM) components. ADAMTS9 gene codes for metalloproteinase involved in ECM degradation20. CSGALNACT1 is involved in chondroitin sulfate biosynthesis, one of the GAGs associated to core protein as proteoglycans that also form the component of ECM21.
ECM has three basic parts that involves basal lamina, interstitial matrix and perineuronal networks. ECM is dynamic, provides structural stability in the form of basement membrane, plays role in signaling and are critical for neuroplasticity22,23. ECM provides an appropriate environment for the development and functioning of cells, like muscle and nerve cells24,25. In the present study, variations in genes encoding collagen and laminins may disrupt basement membrane which has important role in neuroplasticity and maintaining synapses.
Other gene variants observed in the present study are in TMOD2gene involved in muscle contraction, HTR2B in signaling serotonin receptors, RSC1A1 , TRPV2 and WNK1 genes in transport of small molecules in stimuli-sensing channels. All of them are involved in communication between neuron and muscle cells and may coordinate production of speech. At neuro muscular junctions (NMJ), motor neurons release neurotranmitters that bind to post-synaptic receptors, leading to contraction of muscles26. Hence disturbances in signaling system might occur at NMJ that may disrupt speech.
ARSD belongs to cluster of sulfatase genes that hydrolyse GAGs that are found to represent truncated pseudogenes27.SPTBN5 is involved in vesicle transport and NCAM signaling (neural cell adhesion molecule) that plays an important role in nervous system development and synaptic plasticity28.
Thus most of the genes identified form extra cellular matrix molecules, involved in signaling functions indicating overall cell communication deficits. These pathways very well coincide with findings of neuroimaging (deficits in white matter tracts)8,29 and genetic animal studies (deficits in corpus callosum)5which conclude cell communication deficits as the major cause for stuttering. Shugart et al., 30 implicated, interesting candidate genes (on chromosome 18) like desmoglein/desmocolin family and neuronal cadherin 2 gene that helps in cell-cell communication and cell adhesion. Such communications are important in neurons involved in the production of speech. As opposed to traditional thinking that origin of speech and language could be localized to one part of our brain, today neural bases are in fact found in the circuits that connect different regions of brain31. In a more elaborate GWAS study, 10 candidate genes were identified to be strongly associated with stuttering32. Globally the genes implicated in stuttering till date, in all affected individuals were reported to have only one mutant copy with reduced level of functional protein (haploinsufficiency) rather than total absence33.
Our ES approach in two multiplex stuttering families point to interesting putative genes involved in signaling and transport. However in STU-65 family, a homozygous variant in NLRP11 gene and a heterozygous variant in NAGPA gene were also identified. The role of these variants must be explored by confirming its segregation in the other affected members of the family and also through functional validation.
Our investigation is of a descriptive type and the potential candidate genes identified should be viewed with caution because some of the gene variants may not have direct role in the development of the disorder. ES in additional members would further narrow down the variants. Functional studies of the identified variants would help in understanding the biological mechanism. Yet, our results support the fact that stuttering is a polygenic disorder with a complex genetic background and we hypothesize multiple and combined mechanisms to be involved in the genesis of stuttering. The variable expressivity of the disorder in our families could be explained by this genetic heterogeneity.
ES addresses only protein coding regions of genes within genome (1.5%); 80% of DNA that was believed to be junk is now biologically active that makes RNA, of which 20% make functional RNAs involved in regulation (http://jonlieffmd.com/blog/mind-and-molecular-genetics-in-the-neuron-3-evolution). The regulatory regions are 20 times bigger than protein coding regions. Thus the complexity lies not only in the number of genes involved but also may be due to the huge regulatory dynamics.