Supplementary Note. Nature Genetics: doi: /ng.2928

Transcription

1 Supplementary Note Loss of heterozygosity analysis (LOH). We used VCFtools v to extract only singlenucleotide variants with minimum depth of 15X and minimum mapping quality of 20 to create a ped file. SNPs with a call rate of 0.2 were plotted from the ped file in R version using the pheatmap version package. DNA methylation analysis. Global DNA methylation was evaluated using the Infinium HumanMethylation450 BeadChip Array. Briefly, 1 µg of each sample DNA underwent bisulfite conversion using using the EZ DNA methylation kit according to the manufacturer s recommendation for the Illumina Infinium Assay. Bisulfite-treated DNA was then hybridized to arrays according to the manufacturer s protocol. We used GenomeStudio V (Illumina) for methylation data assembly and acquisition as well as background correction and normalization. Methylation levels for each CpG residue are presented as β values, estimating the ratio of the methylated signal intensity over the sum of the methylated and unmethylated intensities at each locus. The average β value reports a methylation signal ranging from 0 to 1 representing completely unmethylated to completely methylated values, respectively. All probes with detection p-values >0.01 were removed. Differential methylation was performed in GenomeStudio by comparing 8 SCCOHT samples to two individual pools of normal fallopian tissue. Resultant β values were assigned a DiffScore to measure statistical significance. All p- values were corrected by calculating a false discovery rate. Probes with DiffScores 13 or -13 and delta β values 0.2 or -0.2 were considered statistically significant and differentially methylated.

2 Figures Supplementary Figure 1. LOH analysis in SCCOHT tumors with SMARCA4 mutations. LOH analysis was performed using bcbio- nextgen 0.7.7a- e91123c to map reads with BWA 0.7.5a to GRCh37 and freebayes v for joint variant calling restricted to chr. 19: 10,667,750 11,554,548. The heat map shows SNPs with a call rate of 0.2. No evidence of LOH was observed. Supplementary Figure 2. Genome view of SMARCA4 450K methylation data. The top panel is a scatter plot displaying differential β values for all 44 SMARCA4 CpG probes in eight SCCOHT samples. Only two probes demonstrate differential hypomethylation (indicated by a red box). The upper limit of the y axis does not exceed 0.11, indicative of a lack of differential hypermethylation for any probe.

3 Supplementary Figure 3. SMARCA4 methylation status in SCCOHT tumors and normal fallopian tube tissues. Vertical scatter plot of the average β values for 44 SMARCA4 450K CpG methylation probes in 8 SCCOHTs and 2 pools of normal fallopian tissue. There is no significant difference in SMARCA4 methylation between normal tissue and SCCOHT.

4 Supplementary Figure 4. SMARCA4 immunohistochemistry in normal premenopausal ovary and fallopian tube. H&E- stained sections (A, C, E) and SMARCA4 immunohistochemistry (B, D, F) demonstrating strong, uniform positive nuclear staining in oocytes (B), granulosa cells of primordial (B) and secondary (D) follicles, theca cells (D) and secretory and ciliated cells throughout the fallopian tube ampulla (F) and fimbria (data not shown). In contrast, the ovarian stroma stains weakly (B) or is negative (data not shown). 400 magnification. Scale bars, 50 μm.

5 Tables Supplementary Table 1. Characteristics of SCCOHT patient samples analyzed by next- generation sequencing. Sample Age at diagnosis (years) FIGO Stage Ethnicity SCCOHT tissue source Hypercalcemia SCCO- 002* 26 IA European Tumor recurrence Yes SCCO IA European Primary tumor Yes SCCO IC European Primary tumor Yes SCCO IIIC European Primary tumor Yes SCCO IIIC African American Tumor recurrence Yes SCCO IIIA African American Primary tumor Yes SCCO IIIC European Primary tumor N/A DAH23 30 IA N/A Primary tumor Yes DAH456* 39 IIIC Hispanic Primary tumor N/A DAH IV European Primary tumor N/A DG1006* 34 N/A N/A Primary tumor Yes DG1219* 37 I European Tumor recurrence Yes *Cases with sequenced Tumor/Normal pair. N/A, information not available.

6 Supplementary Table 3. Immunohistochemistry (IHC) analysis of SCCOHT tumors. Age at diagnosis SMARCA4 mutations Sample SMARCA4 IHC SMARCB1 IHC (years) (protein) SCCO- 002* 26 None Negative Positive SCCO p.arg979* N/A N/A SCCO None Positive Negative SCCO p.gly241fs Negative Positive SCCO None Negative Positive SCCO p.glu667fs p.leu1161fs N/A N/A SCCO p.arg1189* N/A N/A DAH23 30 Splice site mutation Negative N/A DAH456* 39 None Positive N/A DAH p.arg1093* N/A N/A DG1006* 34 p.glu952fs p.ser1591fs Negative N/A DG1219* 37 Splice site mutation Negative N/A BIN- 67 Tumor cell line 2 Splice site mutations Negative Positive SCCO N/A Negative Positive SCCO N/A Negative Positive SCCO N/A Negative Positive SCCO N/A Negative Positive SCCO N/A Negative Positive SCCO N/A Negative Positive SCCO N/A Negative Positive SCCO N/A Positive N/A SCCO N/A Negative N/A *Cases with sequenced Tumor/Normal pair. N/A, sample not available.