Evaluation of methylation profiles of an epidermal growth factor receptor gene in a head and neck squamous cell carcinoma patient group

M Mutlu; P Mutlu; S Azarkan; Ö Bayır; B Öcal; G Saylam; MH Korkmaz

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Evaluation of methylation profiles of an epidermal growth factor receptor gene in a head and neck squamous cell carcinoma patient group

| 23 mar 2021

Balkan Journal of Medical Genetics

Volume 23 (2020): Numero 2 (November 2020)

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Article Category: Original article

Pubblicato online: 23 mar 2021

Pagine: 65 - 72

DOI: https://doi.org/10.2478/bjmg-2020-0025

Parole chiave
Epidermal growth factor receptor () gene, epigenetics, head and neck squamous cell carcinoma (HNSCC), methylation, methylation-specific-polymerase chain reaction (MS-PCR)

© 2020 Mutlu M, Mutlu P, Azarkan S, Bayır Ö, Öcal B, Saylam G, Korkmaz MH, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Amplification curves of methylated reactions from both HNSCC and healthy control groups.* Positive methylation control.

Amplification curves of unmethylated reactions from both HNSCC and healthy control groups.* Positive methylation control.

Polymerase chain reaction primers for methylated and unmethylated DNA sequences for the EGFR gene.

Primer Name	Sequences (5’>3’)	PCR Product
EGFR-M Forward	TGT TTT TTT CGC GTT TCG GTT CGC GC	150 bp
EGFR-M Reverse	CGT CTA AAC GAC GAC GAC CGC CG
EGFR-UM Forward	TGT TTT GTT TTT TTG TGT TTT GGT TTG TGT
EGFR-UM Reverse	CAT CCA ATC TAA ACA ACA ACA ACC ACC A	150 bp

Determination of methylation profile after methylation-specific-polymerase chain reaction.

MS-PCR
Methylated	Unmethylated	Methylation Profile
[–]	[+]	unmethylated
[+]	[–]	methylated
[+]	[+]	methylated
[–]	[–]	excluded from study

Methylation profiles of control and patient groups.

Control Group	MS-PCR				Methylation Profile	Patient Group	MS-PCR				Methylation Profile
	Ct	M	Ct	UM			Ct	M	Ct	UM
Positive Control^a	27.33	[+]	40.00	[–]	M	Positive Control^a	27.33	[+]	40.00	[–]	M
C-1	34.37	[+]	32.28	[+]	M	P-1	33.72	[+]	31.41	[+]	M
C-2	32.86	[+]	30.67	[+]	M	P-2	29.87	[+]	29.77	[+]	M
C-3	31.43	[+]	28.88	[+]	M	P-3	33.21	[+]	31.95	[+]	M
C-4	32.92	[+]	29.35	[+]	M	P-4	31.68	[+]	29.50	[+]	M
C-5	31.82	[+]	29.74	[+]	M	P-5	32.60	[+]	29.89	[+]	M
C-6	31.93	[+]	28.99	[+]	M	P-6	30.82	[+]	29.36	[+]	M
C-7	31.91	[+]	28.57	[+]	M	P-7	32.47	[+]	29.00	[+]	M
C-8	35.95	[–]	32.27	[+]	UM	P-8	31.66	[+]	28.34	[+]	M
C-9	31.25	[+]	29.97	[+]	M	P-9	30.84	[+]	29.53	[+]	M
C-10	29.84	[+]	27.52	[+]	M	P-10	32.65	[+]	29.36	[+]	M
C-11	32.69	[+]	30.40	[+]	M	P-11	35.32	[–]	33.99	[+]	UM
C-12	33.58	[+]	33.50	[+]	M	P-12	36.56	[–]	34.23	[+]	UM
C-13	33.67	[+]	29.72	[+]	M	P-13	32.74	[+]	30.43	[+]	M
C-14	34.55	[+]	30.58	[+]	M	P-14	34.57	[+]	32.49	[+]	M
C-15	33.94	[+]	30.88	[+]	M	P-15	33.73	[+]	30.99	[+]	M
C-16	33.88	[+]	29.60	[+]	M	P-16	35.42	[–]	33.56	[+]	UM
C-17	34.66	[+]	32.72	[+]	M	P-17	35.56	[–]	31.58	[+]	UM
C-18	32.31	[+]	29.43	[+]	M	P-18	34.46	[+]	31.25	[+]	M
C-19	33.84	[+]	30.86	[+]	M	P-19	36.38	[–]	33.45	[+]	UM
C-20	33.76	[+]	30.72	[+]	M	P-20	35.12	[–]	32.46	[+]	UM
C-21	33.30	[+]	29.73	[+]	M	P-21	35.98	[–]	33.72	[+]	UM
C-22	34.56	[+]	29.86	[+]	M	P-22	33.67	[+]	31.98	[+]	M
C-23	33.40	[+]	30.30	[+]	M	P-23	34.28	[+]	31.94	[+]	M
C-24	33.97	[+]	31.45	[+]	M	P-24	34.77	[+]	31.80	[+]	M
C-25	33.30	[+]	30.37	[+]	M	P-25	33.33	[+]	31.58	[+]	M
C-26	32.88	[+]	30.47	[+]	M	P-26	32.55	[+]	30.69	[+]	M
C-27	32.35	[+]	31.44	[+]	M	P-27	33.65	[+]	31.66	[+]	M
C-28	34.17	[+]	31.74	[+]	M	P-28	32.99	[+]	29.57	[+]	M
C-29	33.24	[+]	30.55	[+]	M	P-29	33.22	[+]	28.42	[+]	M
C-30	32.74	[+]	28.52	[+]	M	P-30	35.51	[–]	28.80	[+]	UM
C-31	33.21	[+]	27.67	[+]	M	P-31	33.48	[+]	28.53	[+]	M
C-32	35.24	[+]	30.34	[+]	UM	P-32	34.27	[+]	29.92	[+]	M
C-33	32.86	[–]	27.26	[+]	M	P-33	32.63	[+]	27.98	[+]	M
C-34	32.42	[+]	28.35	[+]	M	P-34	32.88	[+]	27.72	[+]	M
C-35	32.29	[+]	28.39	[+]	M	P-35	33.68	[+]	29.86	[+]	M
C-36	33.80	[+]	28.59	[+]	M	P-36	33.67	[+]	28.82	[+]	M
C-37	34.38	[+]	27.65	[+]	M	P-37	34.92	[+]	30.58	[+]	M
C-38	33.61	[+]	27.66	[+]	M	P-38	33.78	[+]	29.73	[+]	M
C-39	33.81	[+]	28.85	[+]	M	P-39	35.75	[–]	28.32	[+]	UM
C-40	32.49	[+]	28.40	[+]	M	P-40	34.72	[+]	29.49	[+]	M
C-41	33.84	[+]	29.66	[+]	M	P-41	32.83	[+]	28.61	[+]	M
C-42	34.29	[+]	28.94	[+]	M	P-42	35.31	[–]	30.58	[+]	UM
C-43	33.97	[+]	29.58	[+]	M	P-43	32.45	[+]	27.63	[+]	M
C-44	35.82	[–]	30.24	[+]	UM	P-44	33.76	[+]	29.67	[+]	M
C-45	35.75	[–]	30.19	[+]	UM	P-45	32.99	[+]	28.25	[+]	M
C-46	35.90	[–]	30.22	[+]	UM	P-46	33.71	[+]	28.53	[+]	M
C-47	33.00	[+]	28.23	[+]	M	P-47	32.89	[+]	28.45	[+]	M
C-48	33.59	[+]	29.34	[+]	M

Relation of the EGFR gene promoter methylation profile of patient group with age, gender and clinicopathological parameters.

Parameters	n	EGFR Methylation Profile		p Value
		Methylated	Unmethylated
Age:<50>50	443	334	19	0.908^a
Gender:malesfemales	416	334	82	0.494^a
Tumor stage:I-IIIII-IV	2027	1423	64	0.184^a

The EGFR gene promoter methylation profile.

Sample	EGFR Promoter Methylation Profile (%)		p Value
	Methylated	Unmethylated
Control	43 (90.0);	5 (10.0)	0.121^a
Patient	37 (79.0)	10 (21.0)

Clinicopathological parameters of the control and patient groups.

	Control Group (n=48) (%)	Patient Group (n=47) (%)
Gender	males: 36 (75.00); females: 12 (25.00)	males: 41 (87.00); females: 6 (13.00)
Median age	58	59
Tumor type	–	Larynx: 31 (65.96)Hypopharynx: 5 (10.64)Lip: 3 (6.39)Tongue: 2 (4.26)Buccal: 2 (4.26)Auricular: 1 (2.13)Retromolar/oral: 1 (2.13)Paranasal sinus: 1 (2.13)Parotid gland: 1 (2.13)
Tumor stage	–	T1: 10 (21.27)T2: 10 (21.27)T3: 20 (42.55)T4: 7 (14.89)

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Evaluation of methylation profiles of an epidermal growth factor receptor gene in a head and neck squamous cell carcinoma patient group

Article Category: Original article

Pubblicato online: 23 mar 2021

Pagine: 65 - 72

DOI: https://doi.org/10.2478/bjmg-2020-0025

Parole chiave
Epidermal growth factor receptor () gene, epigenetics, head and neck squamous cell carcinoma (HNSCC), methylation, methylation-specific-polymerase chain reaction (MS-PCR)

© 2020 Mutlu M, Mutlu P, Azarkan S, Bayır Ö, Öcal B, Saylam G, Korkmaz MH, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Figure 2

Polymerase chain reaction primers for methylated and unmethylated DNA sequences for the EGFR gene.

Determination of methylation profile after methylation-specific-polymerase chain reaction.

Methylation profiles of control and patient groups.

Relation of the EGFR gene promoter methylation profile of patient group with age, gender and clinicopathological parameters.

The EGFR gene promoter methylation profile.

Clinicopathological parameters of the control and patient groups.

Evaluation of methylation profiles of an epidermal growth factor receptor gene in a head and neck squamous cell carcinoma patient group

Article Category: Original article

Pubblicato online: 23 mar 2021

Pagine: 65 - 72

DOI: https://doi.org/10.2478/bjmg-2020-0025

Parole chiaveEpidermal growth factor receptor () gene, epigenetics, head and neck squamous cell carcinoma (HNSCC), methylation, methylation-specific-polymerase chain reaction (MS-PCR)

© 2020 Mutlu M, Mutlu P, Azarkan S, Bayır Ö, Öcal B, Saylam G, Korkmaz MH, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Figure 2

Polymerase chain reaction primers for methylated and unmethylated DNA sequences for the EGFR gene.

Determination of methylation profile after methylation-specific-polymerase chain reaction.

Methylation profiles of control and patient groups.

Relation of the EGFR gene promoter methylation profile of patient group with age, gender and clinicopathological parameters.

The EGFR gene promoter methylation profile.

Clinicopathological parameters of the control and patient groups.

Parole chiave
Epidermal growth factor receptor () gene, epigenetics, head and neck squamous cell carcinoma (HNSCC), methylation, methylation-specific-polymerase chain reaction (MS-PCR)