	US 11,993,819 B2
	Method of determining a risk of cancer
Anders Skanderup, Singapore (SG); Yu Amanda Guo, Singapore (SG); and Sarah Ng, Singapore (SG)
Assigned to Agency for Science, Technology and Research, Singapore (SG)
Appl. No. 16/977,197
Filed by Agency for Science, Technology and Research, Singapore (SG)
PCT Filed Mar. 1, 2019, PCT No. PCT/SG2019/050119 § 371(c)(1), (2) Date Sep. 1, 2020, PCT Pub. No. WO2019/168478, PCT Pub. Date Jun. 6, 2019.
Claims priority of application No. 10201801692U (SG), filed on Mar. 1, 2018.
Prior Publication US 2021/0155991 A1, May 27, 2021
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/6827 (2018.01)

CPC C12Q 1/6886 (2013.01) [C12Q 1/6827 (2013.01)]

10 Claims

1. A method of detecting, determining and/or diagnosing gastrointestinal cancer in a human subject, the method comprising:

i) extracting nucleic acid from a fluid biological sample of the subject, wherein the fluid biological sample is selected from the group consisting of blood, plasma, serum, and combinations thereof,

ii) contacting the nucleic acid from the sample with an agent for detecting the presence of a mutation in one or more focal regions with high mutation rates,

iii) detecting the presence of mutation in one or more focal regions with high mutation rates,

wherein the one or more focal regions is selected from the group consisting of CTCF-binding sites (CBS) overlapping regions set forth in the table 1 below:


Chromosome	Start site	End site

6	50570094	50570120
8	71000992	71001012
1	209422184	209422222
2	49173770	49173816
4	182064578	182064613
X	104435106	104435140
14	70285576	70285601
6	73122084	73122123
8	70576141	70576184
13	36552821	36552860
3	164903700	164903728

or portions or 1 to 4 bp sequences flanking the 5′ end thereof,

and non-CBS regions set forth in the table 2 below:


Chromosome	Start site	End site

7	68391104	68391132
7	136495924	136495948
2	57627616	57627640
16	8381278	8381302
5	23824204	23824224
7	67614923	67614943
8	65161396	65161420
7	4937707	4937736
12	126996666	126996686
4	5415060	5415082
X	137405623	137405655
3	171164993	171165017
4	144748744	144748764
9	25481736	25481758
2	77150455	77150477
3	104801455	104801477
X	125548690	125548710
14	83046706	83046744

or portions or 1 to 4 bp sequences flanking the 5′ end thereof, and

wherein detecting the presence of mutation in the CBS overlapping region(s), or portions or 1 to 4 bp sequences flanking the 5′ end thereof, comprises contacting the biological sample with a primer having a sequence selected from the group consisting of: SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. SEQ ID NO. 83, SEQ ID NO. 84 and combinations thereof and/or wherein detecting the presence of mutation in the one or more focal regions comprises contacting the biological sample with a primer having a sequence selected from the group consisting of: SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, and combinations thereof; and

iv) detecting, determining, and/or diagnosing the presence of gastrointestinal cancer in the subject based on the presence of mutation in the one or more focal regions.