## 2 Materials and Methods
### Whole-Exome Sequencing and Segregation of Rare Variants through Sanger Sequencing
Several databases, including the 1000 genome project ([[https://www.internationalgenome.org/](https://www.internationalgenome.org/)]([https://www.internationalgenome.org/](https://www.internationalgenome.org/)), accessed on 20 June 2021), dbSNP ([[https://www.ncbi.nlm.nih.gov/snp/](https://www.ncbi.nlm.nih.gov/snp/)]([https://www.ncbi.nlm.nih.gov/snp/](https://www.ncbi.nlm.nih.gov/snp/)), accessed on 20 June 2021), EVS ([[https://evs.gs.washington.edu/EVS/](https://evs.gs.washington.edu/EVS/)]([https://evs.gs.washington.edu/EVS/](https://evs.gs.washington.edu/EVS/)), accessed on 20 June 2021), and gnomAD ([[https://gnomad.broadinstitute.org/](https://gnomad.broadinstitute.org/)]([https://gnomad.broadinstitute.org/](https://gnomad.broadinstitute.org/)), accessed on 20 June 2021), were utilized for filtration and validation of identified genetic variants obtained from WES analysis. The University of California Santa Cruz (UCSC) genome database browser ([[http://genome.ucsc.edu/cgibin/hgGateway](http://genome.ucsc.edu/cgibin/hgGateway)]([http://genome.ucsc.edu/cgibin/hgGateway](http://genome.ucsc.edu/cgibin/hgGateway)), accessed on 20 June 2021) was used to obtain the reference sequence (Table 1). Primer3 Plus ([[http://www.bioinformatics.nl/cgibin/primer3](http://www.bioinformatics.nl/cgibin/primer3) plus/primer3 plus.cgi/]([http://www.bioinformatics.nl/cgibin/primer3](http://www.bioinformatics.nl/cgibin/primer3) plus/primer3 plus.cgi/), accessed on 20 June 2021) was used for designing the primers for PCR amplification (Supplementary Materials Table S1). Subsequent sequencing of genetic DNA was accomplished by the ABI3730 genetic analyzer with BigDye chemistry v3.1. A sequence alignment program, BioEdit version 6.0.7 ([[http://www.mbio.csu.edu/BioEdit/bioedit.htm](http://www.mbio.csu.edu/BioEdit/bioedit.htm)]([http://www.mbio.csu.edu/BioEdit/bioedit.htm](http://www.mbio.csu.edu/BioEdit/bioedit.htm)), accessed on 20 June 2021), was used to align the obtained sequences against the reference sequence. Pathogenicity Prediction and Protein Sequence Alignment  
For performing Insilco evaluation, different pathogenicity detection tools were used, including MutationTaster ([[http://www.mutationster.org/](http://www.mutationster.org/)]([http://www.mutationster.org/](http://www.mutationster.org/)), accessed on 20 June 2021), PROVEN ([[http://provean.jcvi.org/seq_submit.php](http://provean.jcvi.org/seq_submit.php)]([http://provean.jcvi.org/seq_submit.php](http://provean.jcvi.org/seq_submit.php))), PolyPhen2 ([[http://genetics.bwh.harvard.edu/pph2/](http://genetics.bwh.harvard.edu/pph2/)]([http://genetics.bwh.harvard.edu/pph2/](http://genetics.bwh.harvard.edu/pph2/)), accessed on 20 June 2021), FATHMM ([[http://fathmm.biocomppute.org/uk/inherited.html](http://fathmm.biocomppute.org/uk/inherited.html)]([http://fathmm.biocomppute.org/uk/inherited.html](http://fathmm.biocomppute.org/uk/inherited.html)), accessed on 20 June 2021). PROVEAN (SIFT ([[https://sift.bi.astaredu.sg/](https://sift.bi.astaredu.sg/)]([https://sift.bi.astaredu.sg/](https://sift.bi.astaredu.sg/)), accessed on 20 June 2021), VarSome ([[https://varsome.com/](https://varsome.com/)]([https://varsome.com/](https://varsome.com/)), accessed on 20 June 2021), and MutPred2 ([[http://mutpred.mutdb.org/](http://mutpred.mutdb.org/)]([http://mutpred.mutdb.org/](http://mutpred.mutdb.org/)), accessed on 20 June 2021). A sequence alignment programme, BioEdit version 6.0.7 ([[http://www.mbio.ncsu.edu/BioEdit/bioedit.htm](http://www.mbio.ncsu.edu/BioEdit/bioedit.htm)]([http://www.mbio.ncsu.edu/BioEdit/bioedit.htm](http://www.mbio.ncsu.edu/BioEdit/bioedit.htm)), accessed on 20 June 2021), was used to align the sequences against the reference sequence (Table 2).