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.