2.3 Molecular analysis
Whole-exome sequencing (WES) was implemented in Madar Medical Genetics Center, Khorramabad, Lorestan, Iran to identify the subtype of HSPs and the corresponding gene, due to heterogenous nature of the disorder. To this end, the genomic DNA of the probands and their relatives were extracted from peripheral blood based on an established salting out protocol.21 Only the probands’ samples were subjected to WES. For Patient B, the SureSelect Human All Exon V6 Kit (Agilent Technologies Inc., Santa Clara, CA, USA) was used to capture exonic region and paired-end sequencing was carried out on illumine NextSeq (Illumina Inc., San Diego, CA, USA). For patient A, Human Core Exome Kit (Twist Bioscience) was used for this purpose. The Burrows-Wheeler Aligner22was implemented to align the sequence reads to GRC38 human reference genome. GATK HaplotypeCaller23, 24 tool was utilized to call all variants within the target regain and annotation was performed using ANNOVAR. At the next step, all variants with more than 0.01 alle frequency in 1000genome, genomAD exome and GenomAD genome were removed and the remaining variants were prioritized according to bioinformatics predictions, inheritance pattern and clinical information. Once WES data was analyzed, the co-segregation analysis of the disease-associated variant was done for understanding the inheritance pattern of the disorder. For this purpose, specific primers Table 1 were designed using online tools such as primer3 (https://primer3.ut.ee), oligoanalyzer (https://eu.idtdna.com/calc/analyzer) and ensembl databases (https://ensembl.org). After PCR amplification, Sanger sequencing was performed using Applied Biosystems 3500 Genetic Analyzer and the sequences were aligned to the reference genome by Codon Code Aligner (https://www.codoncode.com/aligner).