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).