Method for determining a head and neck squamous cell carcinoma

A method for measuring gene methylation, said method comprising:

• (i) obtaining an isolated biological sample from an individual;
• (ii) purifying genomic DNA from said sample;
• (iii) treating the genomic DNA, with bisulfite;
• (iv) amplifying the genomic DNA, after step (iii) by using primer pairs, said primer pairs allowing the amplification of sequences of the genomic DNA region of genes selected from:
• EPHX3, KIF1A, LRRTM1, FLI1, ITGA4, LINC00599, NTM, PARP15, ZAP70, miR193A, miR296, GP1BB, hTERT and any combination thereof, said sequences comprising CpG and/or CpG islands;
• (v) measuring the methylation level of each of the CpG and/or CpG islands falling in the sequences amplified according to the step (iv) and belonging to the following set of genes:
• a) KIF1A, LRRTM1, FLI1, LINC00599, PARP15, ZAP70, miR193A, miR296, GP1BB, hTERT;
• and/or
• b) EPHX3, KIF1A, LRRTM1, FLI1, ITGA4, LINC00599, NTM, PARP15, ZAP70, miR193A, miR296, GP1BB and hTERT, and
• (vi) detecting hypermethylation in
• the CpG and/or CpG islands falling in the sequence of at least one of the following genes:
• EPHX3, KIF1A, LRRTM1, FLI1, ITGA4, LINC00599, NTM, PARP15, ZAP70 and miR193A and detecting hypomethylation in the miR296, GP1BB and hTERT genes, wherein said hypermethylation and said hypomethylation are compared to a normal mucosa of a healthy individual,
• wherein the CpG and/or CpG islands are selected from:
• a CpG/CpG island falling in the genomic region of PARP15 exon 1 SEQ ID NO:
• 53, wherein the CpG/CpG island is selected from nucleotides in positions:
• 1, 23, 50, 54, 60, 67, 74, 82, 86, 93, 100, 102, 108, 114, 117, 129, 146, 148, 155 and 161 of SEQ ID NO:53 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of ITGA4 exon 2 SEQ ID NO:
• 54, wherein the CpG/CpG island is selected from nucleotides in positions:
• 4, 12, 16, 22, 29, 51, 70, 76, 93, 99, 118, 130, 138 and 142 of SEQ ID NO:54 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of NTM exon 1 SEQ ID NO:
• 55, wherein the CpG/CpG island is selected from nucleotides in positions:
• 5, 17, 19, 38, 40, 42, 57, 69, 72, 81, 103, 110, 118, 126 and 142 of SEQ ID NO:55 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of ZAP70 exon 3 SEQ ID NO:
• 56, wherein the CpG/CpG island is selected from nucleotides in positions:
• 2, 13, 15, 17, 23, 26, 45, 52, 60, 63, 67, 74, 79, 85, 95, 105, 110, 120, 128 and 132 of SEQ ID NO:56 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of MIR193A promoter SEQ ID NO:
• 57, wherein the CpG/CpG island is selected from nucleotides in positions:
• 9, 11, 16, 22, 26, 31, 44, 51, 54, 77, 80, 85, 90, 92, 105, 108, 112, 124, 133, 142, 149, 156, 186, 190 and 204 of SEQ ID NO:57 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of EPHX3 exon 1 SEQ ID NO:
• 58, wherein the CpG/CpG island is selected from nucleotides in positions:
• 3, 6, 14, 19, 21, 24, 32, 48, 50, 59, 62, 65, 67, 74, 88, 90, 116, 122, 132, 134, 137, 144, 161, 164, 175, 181, 184, 186, 195, 199, 202, 205, 210, 212 and 219 of SEQ ID NO:58 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of KIF1A exon 1 SEQ ID NO:
• 59, wherein the CpG/CpG island is selected from nucleotides in positions:
• 2, 6, 9, 11, 13, 26, 31, 40, 42, 47, 52, 57, 63, 65, 72, 76, 78, 81, 84, 88, 96, 107, 110, 114, 126, 132 and 140 of SEQ ID NO:59 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of LINC00599 exon 1 SEQ ID NO:
• 60, wherein the CpG/CpG island is selected from nucleotides in positions:
• 3, 10, 13, 21, 35, 39, 43, 52, 59, 69, 71, 76, 80, 82, 86, 88, 114, 127, 132 and 139 of SEQ ID NO:60 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of FLI1 exon 1 SEQ ID NO:
• 61, wherein the CpG/CpG island is selected from nucleotides in positions:
• 3, 14, 32, 44, 46, 49, 59, 70, 87, 116 and 140 of SEQ ID NO:61 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of GP1BB exon 1 SEQ ID NO:
• 62, wherein the CpG/CpG island is selected from nucleotides in positions:
• 11, 14, 19, 27, 36, 51, 67, 74, 81, 88, 91, 109, 117, 119, 121, 125, 127 and 129 of SEQ ID NO:62 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of MIR296 exon 1 SEQ ID NO:
• 63, wherein the CpG/CpG island is selected from nucleotides in positions:
• 3, 8, 10, 29, 45, 77, 94, 106, 109, 116, 127, 152, 160, 172 and 183 of SEQ ID NO:63 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of LRRTM1 promoter SEQ ID NO:
• 64, wherein the CpG/CpG island is selected from nucleotides in positions:
• 3, 6, 10, 13, 20, 22, 25, 28, 32, 45, 49, 52, 56, 59, 63, 66, 70, 89, 106, 111, 113 and 131 of SEQ ID NO:64 and combination thereof;
• and/or
• a CpG/CpG island falling in the genomic region of TERT (hTERT) promoter SEQ ID NO:
• 65, wherein the CpG/CpG island is selected from nucleotides in positions:
• 5, 14, 23, 77, 94 and 106 of SEQ ID NO:65 and combination thereof.