1. Molecular Diagnosis of Mosaic Overgrowth Syndromes Using a Custom-Designed Next- Generation Sequencing Panel 
Fengqi Chang, Liu Liu, Erica Fang, Guangcheng Zhang, Tiansheng Chen, Kajia Cao, Yanchun Li, Marilyn M. Li 
The Journal of Molecular Diagnostics 
Volume 19, Issue 4, Pages (July 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Schematic representation of functional domains of PIK3CA protein and the distribution of pathogenic variants detected using the overgrowth panel. Most of the variants are located in the kinase domain (14 of 25; 56%), followed by the helical domain (6 of 25; 24%). Numbers in parentheses endicate the number of cases with this variant. RBD, RAS-binding domain.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Next-generation sequencing (NGS) and Sanger sequencing shows PIK3CA p.Y1021C in both affected tissue cultures from the left arm and unaffected tissue cultures from the right arm of patient C-25 with much lower variant allele frequency (VAF) in unaffected tissue cultures. Closed arrows indicate the A>G changes detected by NGS and visualized with Integrative Genomics Viewer; open arrows indicate the A>G changes confirmed by Sanger sequencing. A: Affected tissue, VAF 40%. B: Unaffected tissue, VAF 5%.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Representative Sanger confirmation results demonstrate much higher variant allele frequencies (VAFs) in cultured tissues than in the uncultured tissues. Direct (A) and cultured (B) amniotic fluid from C-1. Uncultured (C) and cultured (D) tissue from C-17.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Prenatal diagnosis of mosaic overgrowth syndrome using the overgrowth panel. A: Integrative Genomics Viewer (IGV) view of PIK3CA p.E542K [variant allele frequency (VAF), <1.0%] in direct amniotic fluid from amnioreduction of patient C-1. B: IGV view of PIK3CA p.E542K (VAF, 38.4%) in cultured amniotic fluid from amnioreduction of patient C-1. C: IGV view of PIK3CA p.H1047L (VAF, 10.0%) in affected tissue from products of conception of patient C-2. D: IGV view of PIK3CA p.H1047L (VAF, 26.1%) in cultured affected tissue from products of conception of patient C-2. Arrows indicate the G to A and A to T nucleotide changes in variants PIK3CA p.E542K and PIK3CA p.H1047L, respectively.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Supplemental Figure S1 General work flow of the overgrowth panel.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Supplemental Figure S2
Median sequencing depth of 17 amplicons that interrogate all known pathogenic variants related to phosphatidylinositol 3-kinase (PI3K)/AKT/ mechanistic target of rapamycin (mTOR)–associated mosaic overgrowth syndromes. The median sequencing depth is approximately ×6000.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8. Supplemental Figure S3
Next-generation sequencing (NGS) and Sanger confirmation results of PIK3CA C.3140A>G, (p.H1047R) in different tissue samples of patient C-4. A: Integrative Genomics Viewer image shows the variant detected in the affected tissue by the overgrowth panel. B: Sanger confirmation results.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

9. Supplemental Figure S4
Sanger sequencing results display PIK3CA p.H1047L in both affected and unaffected tissues from patient C-9 (A) and AKT1 p.E17K in both cultured affected and normal skin from tissues patient C-37 (B).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

10. Supplemental Figure S5
Sanger confirmation of the PIK3CA c.1624G>A, (p.E542K) in patient C-1. A: Cultured amniotic fluid (AF) at second trimester (AF1). B: Cultured AF from the amnioreduction (AF2). C: Direct AF from the amnioreduction (AF2). D: Postnatal blood. E: Maternal blood.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

11. Supplemental Figure S6
Sanger confirmation of PIK3CA c.3140A>T, (p.H1047L) in patient C-2. A: Affected tissue from the products of conception (POCs). B: Cultured affected tissue from the POCs. C: Direct amniotic fluid. D: Normal tissue from the POCs. E: Cultured normal tissue from the POCs. F: Maternal blood.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions