Whole Exome Sequencing for Congenital Lymphedema

Whole exome sequencing was performed on DNA extracted from whole blood using previously described method with the following alterations: the Agilent SureSelectXT Human All Exon V4 kit (Agilent Technologies) was used^{29 (link)}, and sequencing was performed on the HiSeq2500 instrument (Illumina) with 100bp paired-end sequencing. Sequence was aligned to the human reference genome (Hg19) using Burrow-Wheeler Aligner (0.5.11)^{30 (link)}; variants were called with Samtools^{31 (link)} and Genome Analysis Toolkit (v.1.6)^{30 (link)} and annotated with Annovar^{31 (link)}. Variants with a quality score <10 were excluded from analysis. Variant filtering and segregation was performed with in house developed software (https://github.com/brendanofallon/VarViewer). All genes previously associated with congenital lymphedema (CCBE1, FOXC2, FLT4, KIF11, GATA2, GJC2, SOX18, FAT4 and PTPN14) were specifically analyzed and no rare or pathogenic variants were found in any family member despite full sequencing coverage. The reported PIEZO1 variants were confirmed by Sanger sequencing using standard methods. Primer sequences are available upon request.

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Lukacs V., Mathur J., Mao R., Bayrak-Toydemir P., Procter M., Cahalan S.M., Kim H.J., Bandell M., Longo N., Day R.W., Stevenson D.A., Patapoutian A, & Krock B.L. (2015). Impaired PIEZO1 function in patients with a novel autosomal recessive congenital lymphatic dysplasia. Nature Communications, 6, 8329.

Publication 2015

Agilent SureSelectXT Human All Exon V4 kit HiSeq2500

Blood Congenital lymphedema Exon Family member Flt4 Gata2 Genes Genome Genome human Human Pathogenic Primer Ptpn14 Variant

Corresponding Organization :

Other organizations : Scripps Research Institute, Howard Hughes Medical Institute, Genomics Institute of the Novartis Research Foundation, ARUP Laboratories (United States), University of Utah

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Variable analysis

independent variables

The Agilent SureSelectXT Human All Exon V4 kit (Agilent Technologies) was used for whole exome sequencing
Sequencing was performed on the HiSeq2500 instrument (Illumina) with 100bp paired-end sequencing

dependent variables

Variants were called with Samtools and Genome Analysis Toolkit (v.1.6) and annotated with Annovar
Variants with a quality score <10 were excluded from analysis
Variant filtering and segregation was performed with in house developed software

control variables

DNA was extracted from whole blood using previously described method
Sequence was aligned to the human reference genome (Hg19) using Burrow-Wheeler Aligner (0.5.11)

controls

No rare or pathogenic variants were found in any family member in genes previously associated with congenital lymphedema despite full sequencing coverage

Annotations

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