Table S1. Primers used in the current study. (*) Biotinylated labeled primer. The location of primers used in the study of the MLH1 promoter methylation is shown in Figure S1.
Gene / Analysis / Primer name / Forward primer (5'-3') / Reverse primer (5'-3') / Ta (ºC)MLH1
MS-MCA / MLH1C_PCR_ext / TATTTTTGTTTTTATTGGTTGG / TAAATACCAATCAAATTTCTCAA / 50
MLH1C_PCR_int / TGTTTTTATTGGTTGGATATTT / CCAATCAAATTTCTCAACTCTATA / 50
MLH1D_PCR_ext / AGGTATTGAGGTGATTGGTTG / CAATTCTCAATCATCTCTTTAATAACA / 50
MLH1D_PCR_int / GGTGATTGGTTGAAGGTATTTT / ATCATCTCTTTAATAACATTAACTAACC / 50
Promoter bisulfite sequencing / MLH1C_BS / TTTTAAAAAYGAATTAATAGGA / AAATACCAATCAAATTTCTCAA / 50
MLH1D_BS / AAATTTGATTGGTATTTAAGTT / CATTCTCAATCTCTTTAA / 50
Clonal promoter bisulfite sequencing / MLH1C-D_BS / TTTTAAAAAYGAATTAATAGGA / CATTCTCAATCTCTTTAA / 50
Promoter bisulfite pyrosequencing / MLH1C_PCR / GGTATTTTTGTTTTTATTGGTTGGATAT / ACTCTATAAATTACTAAATCTCTT* / 57
MLH1C_Seq / TAAAAAGAATTAATAGGAA / 57
MLH1D_PCR / TTGAGAAATTTGATTGGTATTTAAGTTGT / ACATTAACTAACCCCTAAATAACTTCCCC* / 58
MLH1D1_Seq / TGAAGGGTGGGGTTG / 58
MLH1D2_Seq / GATTGGTTGAAGGTATTTT / 58
Promoter sequencing / MLH1promoter_PCR / AACCCTTTCACCATGCTCTG / CCTCGTGCTCACGTTCTTC / 59
MLH1promoter_Seq1 / TACATGCTCGGGCAGTACCT / 54
MLH1promoter_Seq2 / TGAAGAGAGAGCTGCTCGTG / 54
ASE (SNuPE) / rs179997_PCR_cDNA / CACAATGCAGGCATTAGTTTCTC / AGGTACAGGAATGGGTGTGTG / 59
rs179997_PCR_gDNA / GTTTCAGTCTCAGCCATGAG / ACACATGATTCACGCCACAG / 55
rs179997_snupe / TTCTCGACTAACAGCATTTCCAAAGA / 50
ASE (pyrosequencing) / rs179997_Pyr_cDNA / GCCTCAACCGTGGACAATATTC / GCTACGGTTTTATCCTCACATCCA* / 64
rs179997_Pyr_gDNA / GCCTCAACCGTGGACAATATTC / CGACATACCGACTAACAGCATTTC* / 56
rs179997_Pyr_Seq / GGACAATATTCGCTCC / 50
EPM2AIP1
ASE (SNuPE) / rs9311149_PCR / GTCCTGTTGTAGCAGTGAATAT / GCAGCATTGGAGAATTGGTA / 59
rs9311149_Seq1 / TAGGTCCTTACCAGTTACTG / 54
rs9311149_Seq2 / CATCATTAGGGAAGATCTAG / 54
rs9311149_snupe / TCCTTGAAACACTTGAACACTTGAT / 50
BRAF
BRAF V600E screening (SNuPE) / BRAF_PCR / CCTAAACTCTTCATAATGCTT / ATAGCCTCAATTCTTACCAT / 55
BRAF_snupe / TAAAAATAGGTGATTTTGGTCTAGCTACA / 50
1
Figure S1. Location of primers used in the study of MLH1 promoter methylation. Map of the CpG islands encompassing the MLH1 and EPM2AIP1 promoters (adapted from MethPrimer program). Two CpG islands are identified (in blue), comprising the C and D promoter regions. Each small vertical red line represents a CpG site. Primer position is indicated by squares. Amplified bands covering regions C, D or both are shown as blue, brown and black lines, respectively. The translation start sites of MLH1 and EPM2AIP1 are indicated by +1.
1
Figure S2. Confirmation of the constitutional MLH1 epimutation of case 34. A. Analysis of the MLH1 promoter C and D regions by MS-MCA, bisulfite sequencing (BIS-SEQ)and pyrosequencing (PYROSEQ). Top panel: MS-MCA of MLH1 promoter. In the analysis of C region, unmethylated control (C-) and RKO DNA (methylated control) show single melting peaks at 73ºC and 77ºC, respectively. In D region, unmethylated control and RKO melting peaks temperature are 76ºC and 82ºC, respectively. Analysis by MS-MCA in PBL DNA from the patient 34 (green line) shows the presence of the methylated peak in both regions.Middle panel: Sequence analysis of bisulfite converted DNA. Unmethylated control shows T at each CpG analyzed, consistent with complete modification of the DNA. RKO DNA shows C at each CpG. Patient DNA shows a mixture of T and C at CpG sites, attributable to partial methylation.Bottom panel: Representative pyrograms obtained in the analysis of C and D MLH1 promoter regions in PBL DNA from the patient. The peaks within the shaded area of the pyrogram correspond to the CpG interrogated. Percentage methylation at each site is calculated as the C:T ratio of peak heights (representing methylated:unmethylated cytosine). X-axis represents the nucleotide dispensation order. Y-axis units are arbitrary representing light intensity.B. Transcriptional inactivation of EPM2AIP1 allele. Representative results of the SNuPE analysis at EPM2AIP1 rs9311149 in genomic DNA and cDNA derived from a heterozygous control and the epimutation carrier. Partial transcriptional silencing of the T allele at EPM2AIP1 rs9311149 in the cDNA of the patient was observed.
A
PYROSEQ
B
SUPPLEMENTARY METHODS
Samples
DNA extraction of colorectal mucosa and tumour tissue from paraffin-embedded material was done after enrichment for normal and tumour cells using the QIAmp DNA Mini Kit (Qiagen). Microsatellite instability testing was performed in tumor DNA using the MSI Analysis System (Promega). Genomic DNA was extracted from peripheral blood lymphocytes (PBL) using the FlexiGene DNA kit (Qiagen, Hilden, Germany). Different samples were acquired from the epimutation carrier: skin fibroblasts, peripheral blood lymphocytes, colorectal tumor and normal adjacent mucosa, and sperm. For fibroblast isolation, a skin biopsy was cut into small pieces and digested with 160 U/ml collagenase type 1 (Sigma, St. Louis, MO) and 0.8 U/ml dispase grade 1 (Roche Diagnostics, Penzberg, Germany).1 Fibroblasts were grown with Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen), 10% fetal bovine serum (Gibco, Invitrogen), and penicillin/streptomycin (Gibco, Invitrogen) at 37ºC and 5% CO2. DNA from cultured fibroblasts was extracted using the Gentra Puregene Cell Kit. Sperm was washed twice in 1x SSC/ 1% SDS, then washed in 1x SSC and incubated in 0.2x SSC/ 1% SDS/ 1M 2-mercaptoethanol for 1 hr at room temperature. DNA was extracted from spermatozoa using a standard phenol-chloroform method and ethanol precipitation. Total RNA was extracted from PBL using Trizol reagent (Invitrogen,Carlsbad, CA), according to the manufacturer’s instructions. cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random primers (Invitrogen).
MLH1 promoter methylation analyses
MS-MCA. Bisulfite converted DNA was used in a nested PCR reaction for the amplification of regions C and D of the MLH1 promoter.2 Each promoter region was preamplified using external primers (Table S1). Eighty ng of bisulfite modified DNA was added to MegaMix double solution (Microzone Ltd., UK) containing 2M of each primer. PCR conditions were: 95 ºC at 10 min followed by 15 cycles of 30s at 95 ºC, 30s at 50°C, 30s at 72ºC and a final elongation step at 72 °C for 10min. The nested PCR was carried out in a LightCycler 480 II (Roche) using 1l of amplified MLH1 promoter fragments in 9l of Light Cycler 480 SYBR Green I Master Kit (Roche) containing 0.5M of internal primers. The amplification protocol was: 95ºC for 10min, followed by 40 cycles of 95ºC for 10s, 50°C for 20s, and 72ºC for 25s. Melting curve analysis was performed by heating the PCR products from 60ºC to 98ºC with an increase of 0.2°C/s whereas fluorescence was monitored continuously.
Bisulfite-Sequencing. One μl of bisulfite converted DNA was used in a 10ul-PCR reaction for the amplification of MLH1 promoterregions C and D2usingDouble Megamix (Microzone Ltd., UK) and 0,2μM of primers (Table S1). The cycling program included 10 min at 94ºC, 35 cycles of 30s at 95°C, 30s at 50°C and 30s at 72ºC and final extension at 72ºC for 10 min. PCR products were purified using ExoSAP-it (Affymetrix, Inc.) and sequenced using the amplification primers and BigDye Terminator v.3.1 Sequencing Kit (Applied Biosystems, Carlsbad, CA).
Clonal Bisulfite-Sequencing. One μl of bisulfite modified DNA was amplified in a PCR reaction using EcoStar DNA polymerase (Ecogen, Spain) and 0,3μM of primers (Table S1). PCR products were purified by ExoSAP-it (Affymetrix, Inc.)and cloned into pGEM-T vector (PromegaCorp, Madison, WI). In order to confirm that transformed cells contained the fragment of interest we performed a colony-PCR using M13 primers. Amplification conditions were: 10 min at 94ºC; and 35 cycles of 1min at 94°C, 1min at 55°C, 1min at 72ºC and final extension at 72ºC for 10 min. The PCR products were analyzed by agarose gel electrophoresis. Twenty individual clones were sequenced using M13 primers and BigDye Terminator v.3.1 Sequencing Kit (Applied Biosystems).
Pyrosequencing. In the case of region D, we designed 2 different sequencing primers, the first primer called as “D1” analyzed the first 3 CpGs and the second primer “D2” analyzed the last 5 CpGs in the region of interest. The program used for amplification was: 95°C for 15 min, 35 cycles of 94°C for 1 min, 1 min at the annealing temperature (Table S1), 72°C for 1 min and a final extension at 72°C for 10 min. Five μl of PCR product were evaluated for % methylation using the PyroMark Q96 MD pyrosequencer (Qiagen,Valencia, CA). If the sample failed at more than one site, it was repeated using 10 μl of PCR product. Purification and subsequent processing of the biotinylated single-stranded DNA was performed according to the manufacter's recommendations at the PyroMark Q96 Vacuum Prep Workstation (Qiagen). The pyrosequencing primers were used in a final concentration of 0.3μM. The pyrosequencing reaction was performed using each specific sequencing primer on a PyroMark Q96 MD pyrosequencer system with the Pyromark Gold Q96 reagents kit. The sequences interrogated were GAGYGGATAGYGATTTTTAAYGYGTAAGYGTATATTTTTTTAGGTAG for promoter C region, GATGGYGTAAGTTATAGTTGAAGGAAGAAYGTGAGTAYGAGGTATTGAGGTGATTGGTTGAAGG for promoter “D1” region, and YGTTGAGTATTTAGAYGTTTTTTTGGTTTTTTTGGYGTTAAAATGTYGTTYGTGGTAGGGGTTATT for promoter “D2” region. The relative levels of the C (representing methylated) and T (representing unmethylated) nucleotides at Y positions of target CpGs sites were determined using the Pyro Q-CpG Software (Qiagen). Each sample was run in triplicates. Methylation at each specific CpG was calculated as the mean of all triplicates. Average % methylation of the whole region was calculated as the mean for the 5 CpGs in C region and the 8 CpGs at the D region.
MLH1 allelic expression analyses
SNuPE: PCR flanking the rs1799977 of genomic DNA and cDNA was performed using Double Megamix (Microzone Ltd., UK) and 0.1 μM of primers (Table S1). cDNA amplification conditions were: 5 min at 94ºC, 35 cycles of 1 min at 94°C, 1 min at 59°C and 1 min at 72ºC and final extension at 72ºC for 7 min. For genomic DNA the cycling program included 10 min at 94ºC, 35 cycles of 30s at95°C, 30s at 55°C and 30s at 72ºC and final extension at 72ºC for 10 min. PCR products were purified using ExoSAP-it (Affymetrix, Inc.) and sequenced if necessary using amplification primers and BigDye Terminator v.3.1 Sequencing Kit (Applied Biosystems). The amplified band was analyzed using the ABI PRISM SNaPshot kit (Applied Biosystems) and a specific primer (Table S1). SNaPshot reactions were carried out in a 10μl volume containing SNaPshot Multiplex Ready Reaction Mix, specific primer (0.2μM) and the purified PCR product. The cycling program included 25 cycles of 96°C for 10s, 50°C for 5s and 60ºC for 30s. Extension products were purified with 1U of shrimp alkaline phosphatase (Amersham, UK) for 15 min at 37ºC and 15min at 75ºC. The purified products were run in an ABI Prism 3130 DNA sequencer and analyzed by GeneMapper v4.0 (Applied Biosystems).
Pyrosequencing: Quantitative pyrosequencing assays were designed as previously described.3 PCR and sequencing primers are shown in Table S1. After PCR amplification of genomic DNA and cDNA, products were sequenced on a PyroMark Q24 pyrosequencing instrument (Qiagen, Valencia, CA). A control in which the template was omitted was used to detect background signal. A nucleotide dispensation order of CAGATCTGA was used to interrogate the sequence of interest A/GTCTTTGGAAA. The proportion of A allele versus G alleles of rs1799977 were obtained using PyroMark Q24 AQ software calculations. The mean of triplicates for both DNA and cDNA were calculated for each sample.
To obtain ASE (allele-specific expression) values, both for SNuPE and pyrosequencing, we used the previously described method: cDNA (peak height major allele/ peak height minor allele) / gDNA (peak height major allele/ peak height minor allele). The final ASE value was calculated as the mean of the ASE values obtained for the triplicates studied in each sample.4 ASE values of 1.0 indicate equal levels of expression form both alleles. ASE values <1.0 indicate reduced expression.
REFERENCES
1Serra E, Ars E, Ravella A et al: Somatic NF1 mutational spectrum in benign neurofibromas: mRNA splice defects are common among point mutations. Hum Genet 2001; 108: 416-429.
2Deng G, Peng E, Gum J, Terdiman J, Sleisenger M, Kim YS: Methylation of hMLH1 promoter correlates with the gene silencing with a region-specific manner in colorectal cancer. Br J Cancer 2002; 86: 574-579.
3Kwok CT, Ward RL, HawkinsNJ, Hitchins MP: Detection of allelic imbalance in MLH1 expression by pyrosequencing serves as a tool for the identification of germline defects in Lynch syndrome. Fam Cancer 2010; 9: 345-356.
4Valle L, Serena-Acedo T, Liyanarachchi S et al: Germline allele-specific expression of TGFBR1 confers an increased risk of colorectal cancer. Science 2008; 321: 1361-1365.
1