Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1

Transcription

1 Supporting Online Material for Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1 Nishant Agrawal,* Mitchell J. Frederick, Curtis R. Pickering, Chetan Bettegowda, Kyle Chang, Ryan J. Li, Carole Fakhry, Tong-Xin Xie, Jiexin Zhang, Jing Wang, Nianxiang Zhang, Adel K. El-Naggar, Samar A. Jasser, John N. Weinstein, Lisa Treviño, Jennifer A. Drummond, Donna M. Muzny, Yuanqing Wu, Laura D. Wood, Ralph H. Hruban, William H. Westra, Wayne M. Koch, Joseph A. Califano, Richard A. Gibbs, David Sidransky, Bert Vogelstein, Victor E. Velculescu,* Nickolas Papadopoulos, David A. Wheeler, Kenneth W. Kinzler,* Jeffrey N. Myers* To whom correspondence should be addressed. (N.A.); (V.E.V.); (K.W.K.); (J.N.M.) This PDF file includes: Published 28 July 2011 on Science Express DOI: /science Materials and Methods Figs. S1 to S6 Tables S1 to S8 References

2 Supplementary Materials and Methods Johns Hopkins Component Preparation of Illumina Genomic DNA libraries Fresh-frozen surgically resected tumor and matched blood were obtained from patients under an Institutional Review Board protocol at the Johns Hopkins Hospital. Tumor tissue was analyzed by frozen section histology to estimate neoplastic cellularity. In order to enrich the samples for neoplastic cells, normal tissue was removed from the samples using macrodissection based on the frozen section histology. HPV tumor status was determined for oropharyngeal tumors per standard clinical care using in-situ hybridization. Hybridization was performed using the HPV III Family16 probe set that captures HPV genotypes 16, 18, 33, 35, 45, 51, 52, 56 and 66. Punctate hybridization signals localized to the tumor cell nuclei defined an HPV associated tumor. HPV16-positive controls included an HPV16-positive oropharyngeal cancer and the SiHa and CaSki cell lines. Genomic DNA libraries were prepared following Illumina s (Illumina, San Diego, CA) suggested protocol with the following modifications. (1) 1-3 micrograms (µg) of genomic DNA from tumor or lymphocytes in 100 microliters (µl) of TE was fragmented in a Covaris sonicator (Covaris, Woburn, MA) to a size of bp. To remove fragments shorter than 150bp, DNA was mixed with 25µl of 5X Phusion HF buffer, 416µl of ddh 2 O, and 84µl of NT binding buffer and loaded into NucleoSpin column (cat# , Clontech, Mountain View, CA). The column was centrifuged at g in a desktop centrifuge for 1 min, washed once with 600 µl of wash buffer (NT3 from Clontech), and centrifuged again for 2 min to dry completely. DNA was eluted in 45 µl of elution buffer included in the kit. (2) Purified, fragmented DNA was mixed with 40 µl of H 2 O, 10 µl of End Repair Reaction Buffer, 5 µl of End Repair Enzyme Mix (cat# E6050, NEB, Ipswich, MA). The 100 µl end-repair mixture was incubated at 20 o C for 30 min, purified by a PCR purification kit (Cat # 28104, Qiagen) and

3 eluted with 42 µl of elution buffer (EB). (3) To A-tail, all 42 µl of end-repaired DNA was mixed with 5 µl of 10X da Tailing Reaction Buffer and 3 µl of Klenow (exo-)(cat# E6053, NEB, Ipswich, MA). The 50 µl mixture was incubated at 37 o C for 30 min before DNA was purified with a MinElute PCR purification kit (Cat # 28004, Qiagen). Purified DNA was eluted with 25 µl of 70 o C EB. (4) For adaptor ligation, 25 µl of A-tailed DNA was mixed with 10 µl of PE-adaptor (Illumina), 10 µl of 5X Ligation buffer and 5 µl of Quick T4 DNA ligase (cat# E6056, NEB, Ipswich, MA). The ligation mixture was incubated at 20 o C for 15 min. (5) To purify adaptorligated DNA, 50 µl of ligation mixture from step (4) was mixed with 200 µl of NT buffer and cleaned up by NucleoSpin column. DNA was eluted in 50 µl elution buffer. (6) To obtain an amplified library, ten PCRs of 50 µl each were set up, each including 29 µl of H 2 O, 10 µl of 5X Phusion HF buffer, 1 µl of a dntp mix containing 10 mm of each dntp, 2.5 µl of DMSO, 1 µl of Illumina PE primer #1, 1 µl of Illumina PE primer #2, 0.5 µl of Hotstart Phusion polymerase, and 5 µl of the DNA from step (5). The PCR program used was: 98 o C 2 minute; 6 cycles of 98 o C for 15 seconds, 65 o C for 30 seconds, 72 o C for 30 seconds; and 72 o C for 5 min. To purify the PCR product, 500 µl PCR mixture (from the ten PCR reactions) was mixed with 1000 µl NT buffer from a NucleoSpin Extract II kit and purified as described in step (1). Library DNA was eluted with 70 o C elution buffer and the DNA concentration was estimated by absorption at 260 nm. Exome and targeted subgenomic DNA capture Human exome capture was performed following a protocol from Agilent s SureSelect Paired- End Target Enrichment System (Agilent, Santa Clara, CA) with the following modifications. (1) A hybridization mixture was prepared containing 25 µl of SureSelect Hyb # 1, 1 µl of SureSelect Hyb # 2, 10 µl of SureSelect Hyb # 3, and 13 µl of SureSelect Hyb # 4. (2) 3.4 µl (0.5 µg) of the PE-library DNA described above, 2.5 µl of SureSelect Block #1, 2.5 µl of SureSelect Block #2 and 0.6 µl of Block #3 was loaded into one well in a 384-well Diamond PCR plate (cat# AB- 1111, Thermo-Scientific, Lafayette, CO), sealed with microamp clear adhesive film (cat#

4 ; ABI, Carlsbad, CA) and placed in GeneAmp PCR system 9700 thermocycler (Life Sciences Inc., Carlsbad CA) for 5 minutes at 95 C, then held at 65 C (with the heated lid on). (3) µl of hybridization buffer from step (1) was heated for at least 5 minutes at 65 C in another sealed plate with heated lid on. (4) 5 µl of SureSelect Oligo Capture Library, 1 µl of nuclease-free water, and 1 µl of diluted RNase Block (prepared by diluting RNase Block 1: 1 with nuclease-free water) were mixed and heated at 65 o C for 2 minutes in another sealed 384- well plate. (5) While keeping all reactions at 65 C, 13 µl of Hybridization Buffer from Step (3) was added to the 7 µl of the SureSelect Capture Library Mix from Step (4) and then the entire contents (9 µl) of the library from Step (2). The mixture was slowly pipetted up and down 8 to 10 times. (6) The 384-well plate was sealed tightly and the hybridization mixture was incubated for 24 hours at 65 C with a heated lid. After hybridization, five steps were performed to recover and amplify captured DNA library: (1) Magnetic beads for recovering captured DNA: 50 µl of Dynal MyOne Streptavidin C1 magnetic beads (Cat # , Invitrogen Dynal, AS Oslo, Norway) was placed in a 1.5 ml microfuge tube and vigorously resuspended on a vortex mixer. Beads were washed three times by adding 200 µl of SureSelect Binding buffer, mixed on a vortex for five seconds, and placed in a Dynal magnetic separator to remove the supernatant. After the third wash, beads were resuspended in 200 µl of SureSelect Binding buffer. (2) To bind captured DNA, the entire hybridization mixture described above (29 µl) was transferred directly from the thermocycler to the bead solution and mixed gently; the hybridization mix /bead solution was incubated in an Eppendorf thermomixer at 850rpm for 30 minutes at room temperature. (3) To wash the beads, the supernatant was removed from the beads after applying a Dynal magnetic separator and the beads were resuspended in 500 µl SureSelect Wash Buffer #1 by mixing on a vortex mixer for 5 seconds and incubated for 15 minutes at room temperature. Wash Buffer#1 was then removed from the beads after magnetic separation. The beads were further washed three times, each with 500 µl pre-warmed SureSelect Wash Buffer #2 after incubation at 65 C for 10 minutes.

5 After the final wash, SureSelect Wash Buffer #2 was completely removed. (4) To elute captured DNA, the beads were suspended in 50 µl SureSelect Elution Buffer, vortex-mixed and incubated for 10 minutes at room temperature. The supernatant was removed after magnetic separation, collected in a new 1.5 ml microcentrifuge tube, and mixed with 50 µl of SureSelect Neutralization Buffer. DNA was purified with a Qiagen MinElute column and eluted in 17 µl of 70 o C EB to obtain 15 µl of captured DNA library. (5) The captured DNA library was amplified in the following way: 15 PCR reactions each containing 9.5 µl of H 2 O, 3 µl of 5 x Phusion HF buffer, 0.3 µl of 10 mm dntp, 0.75 µl of DMSO, 0.15 µl of Illumina PE primer #1, 0.15µl of Illumina PE primer #2, 0.15 µl of Hotstart Phusion polymerase, and 1 µl of captured exome library were set up. The PCR program used was: 98 o C for 30 seconds; 14 cycles of 98 o C for 10 seconds, 65 o C for 30 seconds, 72 o C for 30 seconds; and 72 o C for 5 min. To purify PCR products, 225µl PCR mixture (from 15 PCR reactions) was mixed with 450 µl NT buffer from NucleoSpin Extract II kit and purified as described above. The final library DNA was eluted with 30 µl of 70 o C elution buffer and DNA concentration was estimated by OD260 measurement. Somatic mutation identification by massively parallel sequencing Captured DNA libraries were sequenced with the Illumina GAIIx/HiSeq Genome Analyzer, yielding 100 (2 X 50) or 150 (2 X 75) base pairs from the final library fragments. Sequencing reads were analyzed and aligned to human genome hg18 with the Eland algorithm in CASAVA 1.7 software (Illumina). A mismatched base was identified as a mutation only when (i) it was identified by more than three distinct tags; (ii) the number of distinct tags containing a particular mismatched base was at least 10% of the total distinct tags; and (iii) it was not present in >0.5% of the tags in the matched normal sample. SNP search databases included and Evaluation of genes in additional tumors and matched normal controls. For six selected genes that were altered in at least 2 tumors, the coding region was sequenced in a validation set composed of an independent series of additional HNSCCs and matched

6 controls. TP53, NOTCH1, CDKN2A, PIK3CA, FBXW7, and HRAS sequence analysis was performed in 120, 139, 120, 51, 120, and 120 patients, respectively. PCR amplification and Sanger sequencing were performed following protocols described previously (1) using the primers listed in table S8. M.D. Anderson Component DNA isolation from human specimens Fresh-frozen surgically resected tumor and matched non-malignant adjacent tissue were obtained from consented patients treated for HNSCC at the University of Texas M.D. Anderson Cancer Center, under an Institutional Review Board approved protocol. Frozen tissue was embedded in optimal cutting temperature compound and cryosections from the top and middle of specimens were stained with hematoxylin and eosin prior to being evaluated by a pathologist for the presence of > 60 % tumor nuclei content or absence of tumor (i.e., normal). Samples that passed this criterion were sectioned all the way through and washed once in PBS prior to isolating genomic DNA using an ArchivePure DNA purification kit (5Prime, Gaithersburg, MD). Library Construction DNA samples (5ug) were constructed into SOLiD pre-capture libraries according to a modified version of the manufacturer s protocol (Applied Biosystems, Inc.). Briefly, DNA was sheared into fragments approximately 120 bp in size with the Covaris S2 or E210 system as per manufacturer instructions (Covaris, Inc. Woburn, MA). Fragments were processed through DNA End-Repair (NEBNext End-Repair Module; Cat. No. E6050L) and A-tailing (NEBNext da-tailing Module; Cat. No. E6053L), followed by purification using a QIAquick PCR purification kit (Cat. No ). Resulting fragments were ligated with BCM-HGSC-designed Truncated-TA (TrTA) P1 and TA-P2 adapters with the NEB Quick Ligation Kit (Cat. No. M2200L). Solid Phase Reversible Immobilization (SPRI) bead cleanup (Beckman Coulter Genomics, Inc.; Cat. No. A29152) was used to purify the adapted fragments, after which nick translation and Ligation- Mediated PCR LM-PCR was performed using Platinum PCR Supermix HIFi (Invitrogen; Cat.

7 No ) and 6 cycles of amplification. Following bead purification, PCR products were quantified using PicoGreen (Cat. No. P7589) and their size distribution analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500 (Cat. No ). Primer sequences and a complete library construction protocol are available on the Baylor Human Genome Website ( Exome Capture and DNA Sequencing Precapture libraries (2 ug) were hybridized in solution with either NimbleGen SeqCap EZ Exome Probes (~26 Mbs of coding sequence from ~17,000 genes), or a custom designed solution probe Vcrome1, (~43.9 Mbs of coding sequence from ~23,000 genes), according to the manufacturer s protocol with minor revisions. Specifically, hybridization enhancing oligos TrTA-A and SOLiD-B replaced oligos PE-HE1 and PE-HE2 and post-capture LM-PCR was performed using 12 cycles. Capture libraries were quantified using PicoGreen (Cat. No. P7589) and their size distribution analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500 (Cat. No ). Capture efficiency was evaluated by performing a qpcr-based SYBER Green assay (Applied Biosystems; Cat. No ) with built-in controls (RUNX2, PRKG1, SMG1, and NLK). Capture library enrichment was estimated at 7 to 9-fold over background. Captured libraries were further processed for sequencing, with approximately 6-12 Gbs of sequence generated per capture library on either SOLiD V3 or V4 instruments (Applied Biosystems, Inc). A complete capture protocol can be found on the Baylor Human Genome Website ( Alignment, De-duplication, and BAM File Generation SOLiD reads were mapped to the Ensembl release 45 version of the Human NCBI Build 36 using BFAST v0.6.4 using standard parameters. For each sample, individual runs in BAM format were merged together and duplicates were marked in the merged BAM files using Picard v1.22. Duplicate reads were excluded from further analysis. Sample Identity Verification

8 To verify the identity of each BAM file, we compared our sequencing genotypes with highdensity SNP array data (Affymetrix) from The Cancer Genome Atlas research consortium using a customized concordance analysis pipeline. The pipeline applies two genotype calling methods, e-genotyping, which screens raw reads for expected alleles from each read subset. After SNP calling, which uses the BAM file as input, filters duplicate reads and low mapping quality reads to produces a list of SNPs/INDELs in the format of SAMtools pileup. Our concordance metric incorporates allele frequency to reward matches or penalize mismatches between rare alleles. The metric also rewards exact matches or mismatches, and penalizes one allele matches, which are statically more common among unrelated samples. About 40,000 sites from the SNP array fall in the capture design region and contribute to the concordance metric. Samples are judged to be concordant and uncontaminated when they score significantly higher against their own SNP array data than any other patient, and the average of the scores against other samples is significantly lower. A match, by e-genotyping, is 95 +/- 3%, whereas unrelated samples score approximately 70%. For SNP call analysis, a match is 98 +/- 2%, whereas unrelated samples score approximately 70%. Somatic Mutation Calling The aligned reads from whole exome sequencing were prefiltered to remove reads with 3 or more non-reference bases, including insertions or deletions. This removed approximately 1-3% of reads from the BAM file. Base substitution and indels in SOLiD reads observed by pileup (SamTools 0.1.7) were collected and filtered to remove variant bases with SNP quality <100. For any given variant position in the tumor the variant allele frequency must be 15%. In addition, at least one read harboring the variant must have mapping quality=255 (i.e., uniquely mapped), and one variant must be Phred quality 40. Variants were discarded if they were observed only at the ends of reads, in position 38-50, or if they exhibited strand bias. Variants were annotated as somatic mutation if they were not observed in the normal. Putative somatic variants observed less than 5 times, or in which the coverage in the normal sequence was less

9 than 9 were set aside. Greater than 80% of the target bases have sufficient coverage in both tumor and normal exome to make somatic mutation calls. Annotation Variants were annotated using gene structures from the NCBI RefSeq transcript set. Coding base substitutions were classified as missense, nonsense, splice site, or silent. Insertions and deletions were classified as in frame or frame shifting and submitted to the TCGA Data Coordinating Center. Mutation Validation All somatic and LOH variants were validated using a sequencing chemistry different from the discovery chemistry (SOLiD). PCR primers are designed to amplify the mutation target in both the tumor and the normal and the amplification products were sequenced using the AB 3730 Sanger, or 454 pyrosequencing methods. PCR reactions are cleaned using Exo Sap IT, (VWR, Inc.) for Sanger sequencing and by Solid Phase Reversible Immobilization (SPRI) beads (AMPure XP, Beckman Coulter Genomics) for 454 sequencing. For the Sanger-based validation, forward and reverse reads are generated and variants are called using SNP Detector v 3.0 software. Amplicons in which SNPdetector did not call the mutation were visually examined for evidence of the mutant allele. For the 454 sequencing based validation PCR products from tumor and normal samples are made into separate pools of 1000 amplicons. 454 Titanium sequencing libraries are generated and each pool is sequenced in a separate 454 run. Reads that map to their cognate amplicon in the genome reference sequence are realigned to the amplicon reference with crossmatch and the variant coordinate is examined for the presence of the somatic mutation. There must be at least 50 matching reads in tumor and normal to make a variant call although typically there are reads per amplicon. A variant is validated if it is observed in the tumor in at least 5% of the reads and must not be observed at all in the normal. Variants in which the mutant allele frequency was 40% or greater

10 in the original SOLiD sequencing data exhibited an 80% validation rate in Sanger sequencing. Allele frequencies less than 40% validate with decreasing efficiency by Sanger sequence, and must be validated by 454. Statistics For SNP determination, Genomic DNA from 32 tumor and normal sample pairs was isolated as described above and analyzed on Affymetrix SNP6.0 microarrays according to the manufacturers instructions. The Affymetrix SNP 6.0 data was processed and analyzed using commercial software Partek Genomics Suite TM ( and R packages. The probe sets were mapped to human genome assembly HG19. The Genomics Suite utilizes a circular binary segmentation (CBS) algorithm to identify the copy number change break points and the segments. Segment means were estimated and then used to classify copy number changes as deletions, amplifications, or unchanged. All tumor samples were normalized to the file from corresponding normal tissue. For CNV detection, a copy number less than 1.6 or greater than 2.4 was required in order to call a loss or gain, respectively. A minimum of 20 markers, a p-value less than and a signal-to-noise ratio of 0.4 were also required. We assumed a tumor cellularity of 60% in order to set thresholds for copy number change. For identification of focal regions a segment had to be found in at least 3 samples below 1.4 (>1 copy loss) or above 3.2 (>2 copy gain). Images were created using the Integrated Genomics Viewer (2). For LOH, Genomics Suite employs a Hidden Markov Model (HMM) to find LOH regions based on the genotype error and the expected heterozygous frequency at each informative SNP. Analysis was performed on the tumor-normal pairs. All LOH regions had a heterozygous rate less than 1/3, with a maximum probability of 0.999, genomic decay of 0, and genotype error of 0.01.

11 To determine the passenger probability of NOTCH1 mutations, we considered only the Prevalence screen, in which 107 samples were analyzed. We assumed a background mutation prevalence of 2.5*10-6 (10x the observed mutation Prevalence and the Discovery screen) and determined the probability of discovering 24 or more mutations using a binomial distribution among the NOTCH1 nucleotides analyzed. A Bonferroni correction of 6 was applied to the p- value reported in the text, as there were six genes analyzed in the Prevalence screen. Supplementary Figure Legends Fig. S1. Genome-wide CNVs. Copy number changes identified in 42 HNSCC tumor-normal pairs mapped along each chromosome. The widths of the bars are proportional to the number of samples with a CNV in that region. Green indicates gains and red indicates losses. Fig. S2. Genome-wide CNVs. Copy number changes are shown for 42 HNSCC tumor normal pairs mapped along each chromosome. Each row is a unique sample with the ID shown on the left. Green indicates gains and red indicates losses. RefSeq gene IDs are shown along the bottom. Fig. S3. Chromosome 9p region. Copy number changes are shown for 42 HNSCC tumor normal pairs in a selected region of chromosome 9p around an observed focal deletion (see table S7). Each row is a unique sample with the ID shown on the left. Green indicates gains and red indicates losses. RefSeq gene IDs are shown along the bottom. The selected region is also indicated by a red box along the chromosome at the top of the image. Fig. S4. Chromosome 11q region. Copy number changes are shown for 42 HNSCC tumor normal pairs in a selected region of chromosome 11q around an observed focal deletion (see table S7). Each row is a unique sample with the ID shown on the left. Green indicates gains and red indicates losses. RefSeq gene IDs are shown along the bottom. The selected region is also indicated by a red box along the chromosome at the top of the image.

12 Fig. S5. Chromosome 3q region. Copy number changes are shown for 42 HNSCC tumor normal pairs in a selected region of chromosome 3q around an observed focal deletion (see table S7). Each row is a unique sample with the ID shown on the left. Green indicates gains and red indicates losses. RefSeq gene IDs are shown along the bottom. The selected region is also indicated by a red box along the chromosome at the top of the image. Fig. S6. Chromosome 7p region. Copy number changes are shown for 42 HNSCC tumor normal pairs in a selected region of chromosome 7p around an observed focal deletion (see table S7). Each row is a unique sample with the ID shown on the left. Green indicates gains and red indicates losses. RefSeq gene IDs are shown along the bottom. The selected region is also indicated by a red box along the chromosome at the top of the image. References S1. T. Sjoblom et al., Science 314, 268 (2006). S2. J. T. Robinson et al., Nat Biotechnol 29, 24 (2011).

13 table S1. Summary of sequence analysis of HNSCC. A. Summary of sequence analysis from samples analyzed using Illumina GAIIx/HiSeq (Johns Hopkins University). HN01 HN07 HN09 Average Normal Tumor Normal Tumor Normal Tumor Target Regions 165, Bases in Target Region 37,806, Bases Sequenced (after quality filtering) 8,785,561, Bases Mapped to Genome 7,631,091, Bases Mapped to Targeted Region 4,070,573, Average High Quality Coverage Targeted bases with at least 10 reads (%) Known SNPs identified in targeted region 19,474 19,380 17,662 21,456 20,930 19,494 19,168 Somatic mutations identified in targeted region B. Summary of sequence analysis from samples analyzed using SOLid V3/V4 (Human Genome Sequencing Center) Average Normal Tumor Normal Tumor Normal Tumor Target Regions 198, , , , , , ,071 Bases in Target Region 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 Bases Sequenced (after quality filtering) 11,230,359, Bases Mapped to Genome 8,351,527, Bases Mapped to Targeted Region 2,972,839, Average High Quality Coverage Targeted bases with at least 10 reads (%) Known SNPs identified in targeted region 13,318 12,813 13,102 13,108 13,299 13,952 14,034 Somatic mutations identified in targeted region

14 HN11 HN12 HN14 HN16 HN19 Normal Tumor Normal Tumor Normal Tumor Normal Tumor Normal Tumor ,091 18,919 19,174 19,458 19,479 18,236 19,094 18,861 19,633 19, Normal Tumor Normal Tumor Normal Tumor Normal Tumor Normal Tumor 198, , , , , , , , , ,071 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942, ,575 13,318 14,885 15,210 15,116 14,636 12,958 12,607 13,007 13,

15 HN20 HN22 HN24 HN27 HN32 Normal Tumor Normal Tumor Normal Tumor Normal Tumor Normal Tumor ,378 19,304 19,817 19,741 18,307 18,304 19,586 18,519 19,431 19, Normal Tumor Normal Tumor Normal Tumor Normal Tumor Normal Tumor 198, , , , , , , , , ,071 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942,661 35,942, ,294 13,188 13,087 12,973 13,051 13,290 13,587 13,345 13,053 13,

16 HN33 HN35 HN41 HN42 Normal Tumor Normal Tumor Normal Tumor Normal Tumor ,139 20,919 19,873 19,715 20,014 19,996 19,729 19, Normal Tumor Normal Tumor 198, , , ,071 35,942,661 35,942,661 35,942,661 35,942, ,098 13,041 13,519 13,

17 table S2. Confirmed mutations identified in the discovery set. Sample Gene Site Tobacco HPV Stage Transcript Accession Nucleotide (genomic) Nucleotide (cdna) Amino Acid (protein) Mutation Type Sequencing Method 388 AASS OC Y na T4N2cM chr7: t>a c.242a>t p.e81v Missense SOLiD 91 ABAT OC Y na T3N1M0 18 chr16: g>a c.511g>a p.a171t Missense SOLiD HN32PT ABCA1 L Y na T3N2cM0 CCDS chr09: c>a c.1736g>t p.r579l Missense Illumina HN32PT ABCA12 L Y na T3N2cM0 CCDS chr02: c>g c.6659g>c p.r2220t Missense Illumina HN33PT ABCA3 OC Y na T3N1M0 CCDS chr16: t>c c.2123a>g p.q708r Missense Illumina HN22PT ABCA4 L Y na T4aN2bM0 CCDS747.1 chr01: t>g c.235a>c p.n79h Missense Illumina HN22PT ABCA6 L Y na T4aN2bM0 CCDS chr17: c>t c.3992g>a p.r1331k Missense Illumina 388 ABCA6 OC Y na T4N2cM chr17: t>g c.892a>c p.m298l Missense SOLiD 388 ABCA9 OC Y na T4N2cM chr17: c>g c.4447g>c p.e1483q Missense SOLiD 347 ABCB1 OC Y na T2N0M chr7: t>g c.53a>c p.k18t Missense SOLiD HN24PT ABCB8 H Y na T2N2cM0 CCDS chr07: g>a c.816g>a p.m272i Missense Illumina HN22PT ABCD2 L Y na T4aN2bM0 CCDS chr12: c>a c.1501g>t p.v501l Missense Illumina HN09PT ABCD4 OC Y na T3N2bM0 CCDS chr14: c>g c.1314g>c p.w438c Missense Illumina HN27PT ABCD4 OC Y na T1N1M0 CCDS chr14: _ delct c.837_838delag fs Indel Illumina 388 ABHD13 OC Y na T4N2cM chr13: c>t c.712c>t p.l238f Missense SOLiD 578 ABR OC Y na T3N2M0 29 chr17:860826_860828deltca c.2236_2238deltga fs Indel SOLiD 391 ACACB OC Y na T4N2bM0 32 chr12: g>t c.4208g>t p.r1403l Missense SOLiD 266 ACADSB OC Y na T4N2cM0 36 chr10: c>t c.1145c>t p.t382m Missense SOLiD 91 ACE OC Y na T3N1M chr17: g>a c.2005g>a p.e669k Missense SOLiD 478 ACSL6 OC N na T2N2bM chr5: c>t c.215g>a p.r72q Missense SOLiD 367 ACTC1 OC Y na T3N2M0 70 chr15: c>t c.736g>a p.d246n Missense SOLiD HN22PT ADAM12 L Y na T4aN2bM0 CCDS chr10: c>a c.1232g>t p.c411f Missense Illumina HN22PT ADAM18 L Y na T4aN2bM0 CCDS chr08: a>g c.811a>g p.i271v Missense Illumina HN22PT ADAMTS12 L Y na T4aN2bM0 CCDS chr05: c>g p.g1297a Missense Illumina HN33PT ADAMTS14 OC Y na T3N1M0 CCDS chr10: g>a c.974g>a p.r325h Missense Illumina HN22PT ADAMTS5 L Y na T4aN2bM0 CCDS chr21: c>a c.946g>t p.v316l Missense Illumina HN11PT ADAMTSL1 OC Y na T2N0M0 CCDS chr09: g>a c.14g>a p.r5h Missense Illumina HN33PT ADAP1 OC Y na T3N1M0 CCDS chr07:926190c>t c.329g>a p.r110q Missense Illumina 325 ADCY1 OC Y na T2N1M0 107 chr7: c>t c.1682c>t p.p561l Missense SOLiD HN11PT AHNAK OC Y na T2N0M0 CCDS chr11: t>c c.7459a>g p.m2487v Missense Illumina 388 AIM1 OC Y na T4N2cM0 202 chr6: c>g c.1105c>g p.l369v Missense SOLiD 578 AKAP12 OC Y na T3N2M chr6: t>c c.2717t>c p.i906t Missense SOLiD HN09PT ALAS1 OC Y na T3N2bM0 CCDS chr03: t>g c.934t>g p.c312g Missense Illumina HN22PT ALAS2 L Y na T4aN2bM0 CCDS chr24: a>c c.287t>g p.v96g Missense Illumina 367 ALG1 OC Y na T3N2M chr16: c>g c.328c>g p.q110e Missense SOLiD HN12PT AMD1 OC Y na T4aN0M0 CCDS chr6: g>c c.783g>c p.q261h Missense Illumina HN33PT ANGPT1 OC Y na T3N1M0 CCDS chr08: t>c c.410a>g p.q137r Missense Illumina 266 ANGPT2 OC Y na T4N2cM0 285 chr8: a>t c.1217t>a p.l406h Missense SOLiD 43 ANKH OC Y na T4N2bM chr5: c>t c.1042g>a p.v348m Missense SOLiD 388 ANKRD17 OC Y na T4N2cM chr4: g>a c.5933c>t p.s1978l Missense SOLiD 388 ANKS1A OC Y na T4N2cM chr6: c>a c.502c>a p.l168i Missense SOLiD HN42PT APOB OP N + T2N2cM0 CCDS chr2: t>c c.3239a>g p.n1080s Missense Illumina 166 APPL2 OC Y na T3N0M chr12: a>g c.677t>c p.l226s Missense SOLiD 388 AQP8 OC Y na T4N2cM0 343 chr16: a>g c.176a>g p.n59s Missense SOLiD 43 ARF4 OC Y na T4N2bM0 378 chr3: t>c c.181a>g p.i61v Missense SOLiD HN12PT ARFGEF2 OC Y na T4aN0M0 CCDS chr20: c>t c.4918c>t p.r1640x Nonsense Illumina 578 ARHGAP29 OC Y na T3N2M chr1: c>g c.3067g>c p.d1023h Missense SOLiD

18 HN32PT ARHGAP30 L Y na T3N2cM0 CCDS chr01: c>a c.3188g>t p.r1063l Missense Illumina HN22PT ARID1A L Y na T4aN2bM0 CCDS285.1 chr01: g>t c.1876g>t p.e626x Nonsense Illumina 43 ARID2 OC Y na T4N2bM chr12: c>t c.2710c>t p.q904x Nonsense SOLiD 388 ARID5B OC Y na T4N2cM chr10: g>a c.1549g>a p.d517n Missense SOLiD HN32PT ARL13B L Y na T3N2cM0 CCDS chr03: g>t c.217g>t p.g73x Nonsense Illumina HN22PT ARSB L Y na T4aN2bM0 CCDS chr05: delg c.1798delc fs Indel Illumina HN33PT ASH1L OC Y na T3N1M0 CCDS chr01: g>c c.2771c>g p.s924c Missense Illumina HN35PT ATXN2L OC Y na T2N2bM0 CCDS chr16: c>t c.1508c>t p.p503l Missense Illumina 388 B3GNT3 OC Y na T4N2cM chr19: g>t c.234g>t p.q78h Missense SOLiD 347 BAZ1B OC Y na T2N0M chr7: g>a c.3946c>t p.q1316x Nonsense SOLiD 347 BBS9 OC Y na T2N0M chr7: g>t c.1065g>t p.q355h Missense SOLiD HN20PT BCORL1 OP Y + T2N2bM0 CCDS chr24: c>t c.2350c>t p.r784x Nonsense Illumina HN11PT BMS1 OC Y na T2N0M0 CCDS chr10: g>a c.2936g>a p.g979e Missense Illumina HN22PT BRCA2 L Y na T4aN2bM0 CCDS chr13: c>t c.3515c>t p.s1172l Missense Illumina HN32PT BSDC1 L Y na T3N2cM0 CCDS363.1 chr01: g>a c.238c>t p.q80x Nonsense Illumina 139 BTN3A1 OC N na T3N1M chr6: g>a c.1272g>a p.m424i Missense SOLiD 388 BTNL8 OC Y na T4N2cM chr5: t>g c.884t>g p.l295r Missense SOLiD HN16PT BZRAP1 OC Y na T3N2cM0 CCDS chr17: c>t c.4439g>a p.r1480q Missense Illumina HN27PT BZRAP1 OC Y na T1N1M0 CCDS chr17: g>c c.3671c>g p.p1224r Missense Illumina 266 C11orf16 OC Y na T4N2cM chr11: g>t c.1319c>a p.p440q Missense SOLiD 367 C19orf21 OC Y na T3N2M chr19:709639c>t c.1693c>t p.r565w Missense SOLiD 91 C19orf57 OC Y na T3N1M chr19: g>c c.1885c>g p.r629g Missense SOLiD 385 C1orf125 OC N na T4N1M chr1: a>t c.2633a>t p.k878m Missense SOLiD 388 C1orf173 OC Y na T4N2cM chr1: g>c c.2497c>g p.p833a Missense SOLiD 347 C20orf103 OC Y na T2N0M chr20: c>t c.761c>t p.a254v Missense SOLiD 43 C20orf197 OC Y na T4N2bM chr20: c>a c.358c>a p.l120i Missense SOLiD 385 C20orf200 OC N na T4N1M chr20: c>t Missense SOLiD 325 C3orf23 OC Y na T2N1M chr3: c>g c.205c>g p.l69v Missense SOLiD 578 C4orf33 OC Y na T3N2M chr4: g>a c.178g>a p.e60k Missense SOLiD 388 CABIN1 OC Y na T4N2cM chr22: g>c c.891g>c p.r297s Missense SOLiD 478 CACNA1D OC N na T2N2bM0 776 chr3: g>a c.2113g>a p.e705k Missense SOLiD 385 CACNA1D OC N na T4N1M0 776 chr3: c>t c.5830c>t p.p1944s Missense SOLiD 388 CAMP OC Y na T4N2cM0 820 chr3: g>t c.397g>t p.a133s Missense SOLiD 325 CARD10 OC Y na T2N1M chr22: g>a c.2245c>t p.r749w Missense SOLiD 325 CASP8 OC Y na T2N1M0 841 chr2: g>a c.31g>a p.g11r Missense SOLiD 478 CASR OC N na T2N2bM0 846 chr3: g>c IVS6-1C>G Splice site Splice site SOLiD 347 CCDC108 OC Y na T2N0M chr2: c>t c.2133g>a p.m711i Missense SOLiD 388 CCDC60 OC Y na T4N2cM chr12: c>g c.1049c>g p.s350c Missense SOLiD 266 CCR5 OC Y na T4N2cM chr3: g>t c.1022g>t p.r341l Missense SOLiD 91 CCT6A OC Y na T3N1M0 908 chr7: g>c c.574g>c p.e192q Missense SOLiD HN35PT CD101 OC Y na T2N2bM0 CCDS891.1 chr1: t>g c.77t>g p.v26g Missense Illumina HN35PT CD300LF OC Y na T2N2bM0 CCDS chr17: c>t c.164g>a p.r55q Missense Illumina 388 CD80 OC Y na T4N2cM0 941 chr3: g>c c.57c>g p.f19l Missense SOLiD HN22PT CD8B L Y na T4aN2bM0 CCDS chr02: delc c.694delg fs Indel Illumina 478 CDADC1 OC N na T2N2bM chr13: g>a c.1529g>a p.r510h Missense SOLiD 367 CDH16 OC Y na T3N2M chr16: c>t c.257g>a p.r86q Missense SOLiD 91 CDH9 OC Y na T3N1M chr5: a>c c.487t>g p.l163v Missense SOLiD HN11PT CDKN2A OC Y na T2N0M0 CCDS chr09: c>g IVS2-1G>C Splice site Splice site Illumina HN22PT CDKN2A L Y na T4aN2bM0 CCDS chr09: c>t IVS2-1G>A Splice site Splice site Illumina

19 HN27PT CELSR3 OC Y na T1N1M0 CCDS chr03: c>t c.6989g>a p.r2330h Missense Illumina HN33PT CELSR3 OC Y na T3N1M0 CCDS chr03: _ delctgt c.5534_5537delacag fs Indel Illumina 325 CETN1 OC Y na T2N1M chr18:570508c>t c.100c>t p.r34w Missense SOLiD 388 CFDP1 OC Y na T4N2cM chr16: t>a c.287a>t p.q96l Missense SOLiD 578 CHD1L OC Y na T3N2M chr1: t>g c.902t>g p.f301c Missense SOLiD 266 CHD5 OC Y na T4N2cM chr1: c>g c.898g>c p.d300h Missense SOLiD 325 CHODL OC Y na T2N1M chr21: g>t c.406g>t p.e136x Nonsense SOLiD 388 CLCN4 OC Y na T4N2cM chrx: g>a c.1063g>a p.a355t Missense SOLiD 447 CLDN18 OC N na T2N1M chr3: g>t c.750g>t p.e250d Missense SOLiD HN22PT CNGA3 L Y na T4aN2bM0 CCDS chr02: t>c c.1580t>c p.l527p Missense Illumina 478 CNTNAP1 OC N na T2N2bM chr17: g>a c.1923g>a p.m641i Missense SOLiD 478 COL11A1 OC N na T2N2bM chr1: g>c c.3319c>g p.q1107e Missense SOLiD HN35PT COL6A3 OC Y na T2N2bM0 CCDS chr2: c>t c.7741g>a p.v2581i Missense Illumina 478 COQ3 OC N na T2N2bM chr6: c>g c.649g>c p.d217h Missense SOLiD HN22PT CPNE1 L Y na T4aN2bM0 CCDS chr20: g>c c.899c>g p.s300c Missense Illumina HN22PT CPNE4 L Y na T4aN2bM0 CCDS chr03: c>t c.1549g>a p.a517t Missense Illumina HN22PT CR2 L Y na T4aN2bM0 CCDS chr01: g>t c.670g>t p.g224w Missense Illumina HN27PT CR2 OC Y na T1N1M0 CCDS chr01: g>c c.1700g>c p.s567t Missense Illumina 139 CRABP2 OC N na T3N1M chr1: g>a c.178c>t p.r60c Missense SOLiD 91 CREB3L2 OC Y na T3N1M chr7: c>a c.1328g>t p.s443i Missense SOLiD 325 CSMD3 OC Y na T2N1M chr8: c>t c.4973g>a p.r1658q Missense SOLiD 347 CSMD3 OC Y na T2N0M chr8: c>g c.3587g>c p.r1196t Missense SOLiD 367 CUBN OC Y na T3N2M chr10: c>t c.6773g>a p.r2258h Missense SOLiD HN33PT CUL3 OC Y na T3N1M0 CCDS chr02: c>t c.2126g>a p.r709q Missense Illumina 388 CYP24A1 OC Y na T4N2cM chr20: t>a c.1468a>t p.n490y Missense SOLiD 266 CYP26A1 OC Y na T4N2cM chr10: c>t c.571c>t p.r191c Missense SOLiD 43 DCC OC Y na T4N2bM chr18: a>c c.3148a>c p.m1050l Missense SOLiD 347 DCT OC Y na T2N0M chr13: c>t c.248g>a p.r83h Missense SOLiD 347 DDI1 OC Y na T2N0M chr11: c>g c.922c>g p.q308e Missense SOLiD HN11PT DDX17 OC Y na T2N0M0 CCDS chr22: c>g c.757g>c p.d253h Missense Illumina HN27PT DDX56 OC Y na T1N1M0 CCDS chr07: g>a c.428c>t p.s143f Missense Illumina HN27PT DGCR6 OC Y na T1N1M0 CCDS chr22: c>t c.391c>t p.r131c Missense Illumina 139 DGKG OC N na T3N1M chr3: a>g c.872t>c p.m291t Missense SOLiD 347 DHX34 OC Y na T2N0M chr19: c>t c.3227c>t p.a1076v Missense SOLiD HN14PT DNAH3 OC N na T2N2bM0 CCDS chr16: g>t c.10232c>a p.a3411d Missense Illumina HN22PT DNAH5 L Y na T4aN2bM0 CCDS chr05: t>g c.769a>c p.m257l Missense Illumina 43 DOK6 OC Y na T4N2bM chr18: a>t c.712a>t p.m238l Missense SOLiD HN11PT DPPA2 OC Y na T2N0M0 CCDS chr03: g>a c.205c>t p.q69x Nonsense Illumina HN07PT DPPA4 OC Y na T4aN2cM0 CCDS chr03: c>t c.202g>a p.e68k Missense Illumina 258 DPYS OC Y na T4N2bM chr8: c>t c.1469g>a p.r490h Missense SOLiD HN22PT DSCAM L Y na T4aN2bM0 CCDS chr21: c>a c.4635g>t p.e1545d Missense Illumina HN32PT DSCAML1 L Y na T3N2cM0 CCDS chr11: g>t c.2199c>a p.n733k Missense Illumina HN16PT DSEL OC Y na T3N2cM0 CCDS chr18: c>t c.14g>a p.g5e Missense Illumina HN19PT DST OP N + T2N2bM0 CCDS chr06: g>a c.3901c>t p.q1301x Nonsense Illumina HN32PT DUSP10 L Y na T3N2cM0 CCDS chr01: g>a c.748c>t p.q250x Nonsense Illumina HN32PT DUSP28 L Y na T3N2cM0 CCDS chr02: g>t c.133g>t p.v45f Missense Illumina HN24PT DUSP5 H Y na T2N2cM0 CCDS chr10: c>a c.226c>a p.l76m Missense Illumina 91 E2F7 OC Y na T3N1M chr12: t>a c.2156a>t p.n719i Missense SOLiD 367 EFCAB6 OC Y na T3N2M chr22: t>c c.2573a>g p.e858g Missense SOLiD

20 166 EIF4G1 OC Y na T3N0M chr3: g>a c.1240g>a p.d414n Missense SOLiD 388 ELFN2 OC Y na T4N2cM chr22: c>t c.182g>a p.r61q Missense SOLiD 43 ELMO1 OC Y na T4N2bM chr7: g>a c.661c>t p.q221x Nonsense SOLiD HN33PT ELP4 OC Y na T3N1M0 CCDS chr11: t>a c.233t>a p.l78x Nonsense Illumina HN20PT ENPP1 OP Y + T2N2bM0 CCDS chr06: c>a c.1481c>a p.a494d Missense Illumina HN22PT ENPP1 L Y na T4aN2bM0 CCDS chr06: g>a c.2503g>a p.a835t Missense Illumina HN11PT ENPP2 OC Y na T2N0M0 CCDS chr08: c>t c.1118g>a p.r373h Missense Illumina HN14PT EP300 OC N na T2N2bM0 CCDS chr22: g>t c.4154g>t p.c1385f Missense Illumina 43 EPDR1 OC Y na T4N2bM chr7: g>a c.760g>a p.e254k Missense SOLiD 388 EPHA1 OC Y na T4N2cM chr7: g>c c.600c>g p.f200l Missense SOLiD 478 EPHA7 OC N na T2N2bM chr6: c>g c.1591g>c p.d531h Missense SOLiD 166 EPHA7 OC Y na T3N0M chr6: c>a c.1966g>t p.g656w Missense SOLiD 388 EPHA7 OC Y na T4N2cM chr6: c>g c.391g>c p.d131h Missense SOLiD HN42PT EPHB3 OP N + T2N2cM0 CCDS chr3: g>t c.2101g>t p.v701f Missense Illumina 325 EPS8 OC Y na T2N1M chr12: a>g c.2215t>c p.f739l Missense SOLiD HN16PT ERBB2 OC Y na T3N2cM0 CCDS chr17: a>g c.274a>g p.r92g Missense Illumina HN22PT ERBB4 L Y na T4aN2bM0 CCDS chr02: g>a c.980c>t p.t327i Missense Illumina 166 ETV6 OC Y na T3N0M chr12: g>c c.650g>c p.r217t Missense SOLiD 388 EVI1 OC Y na T4N2cM chr3: c>a c.3130g>t p.a1044s Missense SOLiD HN33PT EXOC4 OC Y na T3N1M0 CCDS chr07: a>g c.1984a>g p.k662e Missense Illumina 325 EXOSC10 OC Y na T2N1M chr1: g>c c.1584c>g p.y528x Nonsense SOLiD 388 F13B OC Y na T4N2cM chr1: c>a c.362g>t p.g121v Missense SOLiD 166 FAM134C OC Y na T3N0M chr17: c>t c.731g>a p.r244h Missense SOLiD 388 FAM179B OC Y na T4N2cM chr14: g>a c.4123g>a p.a1375t Missense SOLiD 43 FAM5B OC Y na T4N2bM chr1: g>a c.1571g>a p.r524h Missense SOLiD 325 FARP2 OC Y na T2N1M chr2: g>a c.2635g>a p.e879k Missense SOLiD HN24PT FAT2 H Y na T2N2cM0 CCDS chr05: g>t c.3140c>a p.s1047x Nonsense Illumina HN14PT FAT4 OC N na T2N2bM0 CCDS chr04: c>g c.3659c>g p.s1220x Nonsense Illumina HN22PT FAT4 L Y na T4aN2bM0 CCDS chr04: c>a c.3178c>a p.r1060s Missense Illumina 266 FBLN5 OC Y na T4N2cM chr14: g>c c.747c>g p.d249e Missense SOLiD 266 FBXO42 OC Y na T4N2cM chr1: g>c c.500c>g p.s167x Nonsense SOLiD HN33PT FBXW7 OC Y na T3N1M0 CCDS chr04: _ inst c.1105_1106insa fs Indel Illumina 166 FBXW7 OC Y na T3N0M chr4: g>c c.1513c>g p.r505g Missense SOLiD 447 FGB OC N na T2N1M chr4: c>t c.1112c>t p.s371l Missense SOLiD HN22PT FGD2 L Y na T4aN2bM0 CCDS chr06: delg c.355delg fs Indel Illumina 367 FGFR2 OC Y na T3N2M chr10: a>g c.1175t>c p.v392a Missense SOLiD HN12PT FLG OC Y na T4aN0M0 CCDS chr1: c>g c.5135g>c p.r1712p Missense Illumina HN14PT FLG OC N na T2N2bM0 CCDS chr01: g>c c.1780c>g p.q594e Missense Illumina HN22PT FLG L Y na T4aN2bM0 CCDS chr01: g>t c.3968c>a p.a1323e Missense Illumina HN32PT FLG2 L Y na T3N2cM0 CCDS chr01: c>t c.482g>a p.r161k Missense Illumina 388 FOXJ2 OC Y na T4N2cM chr12: g>c c.169g>c p.d57h Missense SOLiD 388 FOXJ2 OC Y na T4N2cM chr12: g>c c.258g>c p.k86n Missense SOLiD HN22PT FOXK1 L Y na T4aN2bM0 CCDS chr07: g>a c.986g>a p.s329n Missense Illumina HN22PT FSTL5 L Y na T4aN2bM0 CCDS chr04: c>a c.1847g>t p.g616v Missense Illumina 388 FTSJD2 OC Y na T4N2cM chr6: c>a c.1736c>a p.s579y Missense SOLiD HN22PT GABRA4 L Y na T4aN2bM0 CCDS chr04: t>c c.107a>g p.q36r Missense Illumina HN32PT GABRB3 L Y na T3N2cM0 CCDS chr15: c>a c.730g>t p.g244x Nonsense Illumina 347 GALNT12 OC Y na T2N0M chr9: c>a c.1563c>a p.c521x Nonsense SOLiD 91 GALNT14 OC Y na T3N1M chr2: g>a c.1460c>t p.p487l Missense SOLiD

21 385 GALNT14 OC N na T4N1M chr2: c>t c.976g>a p.v326i Missense SOLiD 391 GC OC Y na T4N2bM chr4: g>c c.1142c>g p.s381x Nonsense SOLiD HN41PT GH2 OP Y + T2N2bM0 CCDS chr17: g>t c.375c>a p.g209r Missense Illumina HN41PT GH2 OP Y + T2N2bM0 CCDS chr17: c>t c.625g>a p.n125k Missense Illumina 347 GIMAP6 OC Y na T2N0M chr7: c>t c.434g>a p.r145h Missense SOLiD 388 GNAI1 OC Y na T4N2cM chr7: g>c c.865g>c p.e289q Missense SOLiD 388 GPC5 OC Y na T4N2cM chr13: g>a c.91g>a p.e31k Missense SOLiD 388 GPR107 OC Y na T4N2cM chr9: a>g c.1515a>g p.i505m Missense SOLiD HN12PT GPR115 OC Y na T4aN0M0 CCDS chr6: c>g c.1608c>g p.i536m Missense Illumina 166 GPR20 OC Y na T3N0M chr8: c>t c.326g>a p.r109h Missense SOLiD HN32PT GPR32 L Y na T3N2cM0 CCDS chr19: g>a c.7g>a p.g3r Missense Illumina 139 GPR63 OC N na T3N1M chr6: c>a c.405g>t p.m135i Missense SOLiD 388 GRAP2 OC Y na T4N2cM chr22: c>t c.614c>t p.p205l Missense SOLiD 266 GRB14 OC Y na T4N2cM chr2: a>g c.233t>c p.i78t Missense SOLiD HN16PT GRID1 OC Y na T3N2cM0 CCDS chr10: g>a c.2702c>t p.s901l Missense Illumina HN32PT GRID1 L Y na T3N2cM0 CCDS chr10: c>a c.2559g>t p.l853f Missense Illumina 367 GRM4 OC Y na T3N2M chr6: g>a c.130c>t p.r44c Missense SOLiD 367 GUF1 OC Y na T3N2M chr4: c>t c.863c>t p.s288f Missense SOLiD 367 HCRTR2 OC Y na T3N2M chr6: c>g c.286c>g p.l96v Missense SOLiD 578 HDAC6 OC Y na T3N2M chrx: a>t c.619a>t p.m207l Missense SOLiD 139 HDC OC N na T3N1M chr15: c>t c.1780g>a p.a594t Missense SOLiD 578 HDX OC Y na T3N2M chrx: t>c c.330a>g p.i110m Missense SOLiD HN11PT HEPH OC Y na T2N0M0 CCDS chr24: a>t c.2383a>t p.r795w Missense Illumina 388 HERC2 OC Y na T4N2cM chr15: g>t c.8424c>a p.c2808x Nonsense SOLiD 385 HEXA OC N na T4N1M chr15: t>c c.856a>g p.t286a Missense SOLiD HN33PT HHIP OC Y na T3N1M0 CCDS chr04: g>a c.1703g>a p.s568n Missense Illumina 578 HIF3A OC Y na T3N2M chr19: g>a c.1396g>a p.g466r Missense SOLiD 388 HIST1H2AK OC Y na T4N2cM chr6: c>g c.366g>c p.e122d Missense SOLiD HN32PT HMG20A L Y na T3N2cM0 CCDS chr15: c>t c.125c>t p.a42v Missense Illumina 367 HN1L OC Y na T3N2M chr16: c>g c.348c>g p.f116l Missense SOLiD 388 HNRNPR OC Y na T4N2cM chr1: c>g c.1505g>c p.r502t Missense SOLiD HN32PT HOXC12 L Y na T3N2cM0 CCDS chr12: c>t c.109c>t p.p37s Missense Illumina HN27PT HOXC6 OC Y na T1N1M0 CCDS chr12: g>a c.413g>a p.g138e Missense Illumina HN11PT HRAS OC Y na T2N0M0 CCDS chr11:523874t>a c.182a>t p.q61l Missense Illumina HN12PT HRAS OC Y na T4aN0M0 CCDS chr11:524286c>g c.37g>c p.g13r Missense Illumina 166 HRAS OC Y na T3N0M chr11:524288c>t c.35g>a p.g12d Missense SOLiD HN11PT HSD17B6 OC Y na T2N0M0 CCDS chr12: t>a c.888t>a p.y296x Nonsense Illumina 388 HTR2A OC Y na T4N2cM chr13: t>c c.161a>g p.n54s Missense SOLiD HN12PT HYDIN OC Y na T4aN0M0 CCDS chr16: g>t c.5893c>a p.l1965m Missense Illumina 325 HYLS1 OC Y na T2N1M chr11: a>c c.27a>c p.q9h Missense SOLiD HN07PT IDS OC Y na T4aN2cM0 CCDS chr24: g>a c.1232c>t p.a411v Missense Illumina HN07PT IDS OC Y na T4aN2cM0 CCDS chr24: c>a c.1231g>t p.a411s Missense Illumina 325 IFNGR1 OC Y na T2N1M chr6: g>c c.1136c>g p.s379c Missense SOLiD 388 IKBKAP OC Y na T4N2cM chr9: t>a c.3685a>t p.t1229s Missense SOLiD 578 IL13RA2 OC Y na T3N2M chrx: c>t c.1123g>a p.e375k Missense SOLiD HN22PT INSC L Y na T4aN2bM0 CCDS chr11: g>t c.1395g>t p.m465i Missense Illumina HN27PT INSC OC Y na T1N1M0 CCDS chr11: t>a c.1709t>a p.l570h Missense Illumina HN12PT INTS3 OC Y na T4aN0M0 CCDS chr1: c>g c.2377c>g p.l793v Missense Illumina HN11PT ITGB1 OC Y na T2N0M0 CCDS chr10: t>a c.2266a>t p.i756l Missense Illumina