Materials and Methods
Both sisters underwent AVR for AS and distal bypass surgeries for
chronic limb-threatening ischemia. As they presented with the typical
features of CALJA, both patients underwent a genetic examination for
definitive diagnosis of CALJA.
Genomic DNA was extracted from whole blood cells using a QIAamp DNA
Blood Mini Kit (QIAGEN, Hilden, Germany). We performed whole exome
sequencing analysis as previously described.8 In
brief, we used the SureSelect Human All Exon V6 kit (Agilent Technology,
Santa Clara, CA) for capture and a HiSeq2500 (Illumina, San Diego, CA)
for sequencing. Reads were aligned to GRC37 using Burrows-Wheeler
Aligner (http://bio-bwa.sourceforge.net/). Variants were identified
using the GATK Unified Genotyper and ANNOVA (http://
annover.openbioinfomatics.org/en/latest/).
This genetic analysis of the patients was approved by the Ethics
Committee of Yamagata University. Both patients and their family members
provided informed consent for clinical and genetic examinations and for
the publication of this report.