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.