Mutational analysis of GNPTAB, GNPTG and NAGPAgene variants
The 12 specific exons spanning across the three genes viz.,GNPTAB , GNPTG and NAGPA implicated in stuttering were screened (Figure 1). Primer sequences were adapted from Kanget al., (2010), after improvising (NCBI’s Primer-BLAST) the sequence coverage of exon 10 of NAGPA gene.
The amplified PCR products were purified by the FavorPrepTM PCR purification kit (FAVOURGEN, Taiwan). The amplicons were sequenced using ABI Prism Big-Dye Terminator 3.1 cycle sequence reaction kit on ABI 3730XL automated sequencer (Applied Biosystems, USA). Chromatograms were analyzed using NCBI nucleotide BLAST (www.ncbi.nlm.nih.gov/) and UCSC genome browser BLAT (genome.ucsc.edu).
Variants identified were predicted using VarSome (https://varsome.com/; tools include various predictors like DANN, Mutation taster, Likelihood Ratio Test - LRT, Mutation assessor, SIFT, Provean etc) and Polyphen tool, to deduce the pathogenicity. Cosegregation of the pathogenic variants among the family members was also evaluated. Novelty and frequency of the variations were compared with ExAC database.
To study the impact of a de novo heterozygous variant(c.802A>C/+) in GNPTG gene identified in one family (STU 63), mRNA expression profile and lysosomal enzyme study was performed along with mucolipidosis screening test. Since the proband alone was affected, while his father, mother and sister served as controls. The plasma collected from fresh blood (5 ml) was used to study the enzyme activity. RNA was isolated using mirVana miRNA isolation kit (Invitrogen, USA) as per manufacturer’s instructions and checked for integrity and purity.
A two step qRT-PCR was used to measure the transcript levels of the mRNAs of interest.
  1. From 500ng of total RNA, cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to manufacturer’s instruction in ABI GeneAmp 9700 PCR System. Reverse transcription reaction mix of 20μl was prepared and was loaded on to ABI GeneAmp 9700 PCR System.
  2. For quantifying gene expression real-time Quantitative PCR was performed on QuantStudio3 Real-Time PCR System using GoTaq DNA polymerase (Promega) in the presence of SYBR Green. The primers specific for the transcripts of GNPTAB, GNPTG and NAGPA (table A2) were designed with the aid of IDT software and checked for specificity with NCBI’s primer-blast. The annealing temperature of the primers were optimized using temperature gradient PCR. Reaction mix for the samples under investigation along with NTC (no template control) was prepared in triplicates for GNPTAB , GNPTG , NAGPAand β-actin genes. The reaction mix was loaded on to a 96 well plate and sealed with MicroAmp® Optical Adhesive Film (Applied Biosystems). Ct value (cycle threshold) is the number of PCR cycles required to achieve a given level of fluorescence. Since Ct value is proportional to logarithm of initial amount of the target, the relative concentration of one target with another is reflected as a difference in cycle number (ΔCt) that is necessary to achieve equivalent level of fluorescence. The expression levels of GNPTAB, GNPTG and NAGPA were measured by relative quantification using the ΔCT method with β-actin as endogenous control.Statistical analysis: Delta Ct values were normalized to the housekeeping β-actin gene for each of the target gene (GNPTAB ,GNPTG and NAGPA ). Statistical analysis was performed by averaging the control Delta Ct values and comparing it with the respective gene expression. Lysosomal targeting of proteins was studied using specific substrate for Arylsulphatase A, Hexosaminidase A and β galactosidase enzymes, with plasma samples from stuttering proband and controls. All members in the family were also evaluated for ML phenotype using a rapid calorimetric screening method.