Additional file 1 of A novel hotspot and rare somatic mutation p.A138V, at TP53 is associated with poor survival of pancreatic ductal and periampullary adenocarcinoma patients
Creators: name: Gourab Saha
name: Singh, Richa
name: Argha Mandal
name: Das, Subrata
name: Esita Chattopadhyay
name: Prasun Panja
name: Paromita Roy
name: Navonil DeSarkar
name: Gulati, Sumit
name: Supriyo Ghatak
name: Shibajyoti Ghosh
name: Sudeep Banerjee
name: Bidyut Roy
name: Saurabh Ghosh
name: Chaudhuri, Dipankar
name: Arora, Neeraj
name: Nidhan K. Biswas
name: Sikdar, Nilabja
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Fig. 1. Hematoxilin and Eosin (H&amp;E) staining of types of tumors. a. H&amp;E staining of a PDAC tumor (left 10x magnification, right 20x magnification). b. H&amp;E staining of an intestinal type of PAC tumor (left 10x magnification, right 20x magnification). c. H&amp;E staining of a pancreatobiliary type of PAC tumor (left 10x magnification, right 20x magnification). Fig. 2. Validation of selected variants. Few of the somatic mutations identified in NGS study are being validated by Sanger sequencing method. Validated nucleotides are marked by vertical lines, in each pair upper one is for tumor DNA and lower one is for normal tissue or blood DNA. Fig. 3. Signature of somatic mutations identified in total patient cohort (n = 93). a. In the X axis different types of mutations is given and Y axis denoting frequency of types of mutations. b. Transition and transversion ration of all point mutations. c. In X axis six different signatures of point mutations is given and Y axis denoting the frequency of different signatures. Fig. 4. Frequency of mutations in other recurrently mutated genes in all patients. Different functional domains of proteins are indicated by different colours. The Y axis denotes frequency of mutations and X axis denotes mutations in different positions of the protein domains. Each vertical bar indicates mutation . Recurrent mutations are marked with red circle a.KRAS mutations.b. SMAD4 mutations. c. CTNNB1 mutations. Fig. 5. Damaging and pathogenic variants of non synonymous mutations (n = 57). Damaging and pathogenic variants of non synonymous mutations (n = 57). The dark ass coloured boxes indicates mutations identified as damaging by different functional prediction tools (Provean, SIFT, and Mutation Assessor). The white coloured boxes mean the tolerated mutations whereas light grey coloured boxes mean “unable to identify”. All the dark grey coloured boxes in the ClinVar_Status row indicate pathogenic or likely pathogenic variants and black coloured boxes are reported as uncertain significance in ClinVar database. Fig. 6. Comparison of reported and novel variants observed in this study with those reported in TCGA and Cosmic database. These are the total mutations identified by both NGS and Sanger sequencing method. The blue coloured boxes in sample row are PAC mixed samples, Sky coloured boxes are PAC intestinal samples, Grey coloured boxes are PAC pancreatobiliary samples and yellow coloured boxes are PDAC samples. The red colouredboxes in COSMIC and TCGA row are the reported mutation in these databases. The boxes marked with “×” in TCGA_PDAC row are not compared as these are PAC mutations data. Among the PDAC samples, red coloured boxes are the mutations which were also observed in TCGA_PDAC (n = 185) mutation data. Fig. 7. Detection of KRAS and TP53 p.A138V mutations by allele specific PCR (a-f). Fig. 8.KRAS 12th codon mutation detection by PCR-RFLP method. Lane 1 represent 100 bp DNA ladder. Lane 2 to 11 represents tumour normal paired samples. Mutant samples can be differentiate with presence of 197 fragments in T1, T2, T3, and T5 as BstNI cannot digest mutant containing fragment. Here sample T4-N4 pair is negative control and T5-N5 pair is positive control of NGS cohort. Fig. 9.KRAS mutation screening by different methods in PDAC samples. First row indicates KRAS mutation status in 36 PDAC samples. The yellow colour indicates G12D mutations, violate colour indicates G12V mutation, sky colour indicates Q61H mutation, green colour indicates G12A mutation, no colour boxes indicate samples with no mutation, and light grey colour indicates samples with failed amplification or poor sequence quality. Other rows indicate different methods (Sanger sequencing, ASPCR, and PCR-RFLP) used for KRAS mutation detection. In the methods rows, red colour indicates identification of mutation, white colour indicates mutations could not be identified, and dark ash colour indicates not applicable due to PCR failure for those samples. Fig. 10. Identification of p.A138V mutation in TP53 gene by Sanger sequencing. The p.A138V mutation in TP53 identified in 16 patients. Here showing two chromatogram (a&amp;b) for p.A138V (Vertical line indicates C/T heterozygous peak) variant identified by Sanger sequencing in 2 patients in the tumour but absent in corresponding normal. Fig. 11. Comparison SSE plots between wild type TP53and A138Vmut TP53. Fig. 12. Intermolecular hydrogen bonding analysis between TP53 (DBD) and DNA complex with respect to wild type and A138Vmut TP53 protein. Number of intermolecular hydrogen bonds between TP53(DBD) (wild type and mutant) and DNA represented in a multivariate plot which shows greater density of data points at 8–12 in wild type (left) and in 5–10 for the mutant (right). Therefore, greater number of intermolecular hydrogen bonds prevailed in the wild type DBD-DNA dynamics simulation than in the corresponding mutant DBD-DNA. Fig. 13. Copy number variation plot for ERBB2 in 93 patients. “X” axis denotes two groups of tissues (Normal and Tumour) of all patients whereas “Y” axis indicates fold change (2-ΔCT) of respective groups. Fig. 14.KRAS hotspot region of 8 tumor samples studied by NGS. Showing reads of KRAS gene focusing on hotspot codon 12 of 8 samples (a-h). Integrative Genomic Browser (IGV) was used for visualization of reads. In figures,“a-h” except “e”, only “C” allele is present in the particular position of KRAS gene, whereas figure“e” indicated “C” and mutant “G” alleles in same position. In figure e the variant allele “G” is represented by orange coloured on the reads. Fig. 15. Comparison of overall survival of p.A138V mutant vs. other TP53 hotspot mutants of TCGA PDAC cohort. Kaplan-Meier survival analysis of all TP53 hotspot mutants of TCGA PDAC cohortalong with TP53 A138V mutants of our patient.