Clinicopathological relevance of BRAF and SMO mutations in Chinese patients with ameloblastoma

Figures & data

Table 1. Primer sequence of BRAF gene for DHPLC and Sanger sequencing.

Exon	Sequence (5′->3′)	Product length	Tm (°C)
BRAF M primer	GGTGATTTTGGTCTAGCTACATA	126	54
	GGCCAAAAATTTAATCAGTGG
TBXAS 1 primer	GCCCGACATTCTGCAAGTCC	100	54
	GGTGTTGCCGGGAAGGGTT
BRAF N primer	ATGCTTGCTCTGATAGGAAAATG	159	58
	AGACAACTGTTCAAACTGATGGG

Note: BRAF M primer and TBXAS 1 primer: DHPLC detecting, TBXAS1 as reference gene. BRAF N primer: Sanger sequencing.

DHPLC, denaturing high-performance liquid chromatography.

Table 2. Primer sequence of SMO gene.

Exon	Sequence (5′->3′)	Product length	Tm (°C)
1	TTTGCTGAGTTGGCGGGGTT	455	58
	CGCCCTCTCCAAAACTCAGG
2	CGTCTGTGGGTCAGAGTGAG	366	58
	CCAGGAGACAAAGCTCTGGG
3	TACCTTTCCCTAAACAAGAGGCT	398	58
	TTCTGATCATGACCCTTCCCTG
4	AGGGTCATGATCAGAAGGGTC	397	58
	AGTATGCAGTAGGGCAGAGCC
5	CTGACTTCTGGGAACCTCCAG	411	58
	GACAGAAGGTGGGTTACTGGC
6	GTGGCGCAGGTATAGTGACTG	348	60
	GCCCTATAGGAGCTAGCTGGG
7	GACTCCAGAGCCTTAGGACCC	304	58
	TCCCTATGGCTAACTTGTCCC
8	AAGCAGTTCTTGGACTGAGCC	382	58
	CCATCCATTGAATCTGCTGTC
9	AGTTGGAAGCTGCAGTGGG	416	58
	CAAGGCTGTGCTAGAGGCAG
10	CTCTGGAAAGAATGGCATCG	336	60
	TTCCAAATAATCTGTGTGCCC
11	AATGGCACTGACTATGGGAGG	344	58
	CCACTCTTCAGATCCTCTGGG
12a	TGCCCCCAGAGGAACAAG	813	58
	ACATAGGCTAGGCTGCCTCC
12b	AACGGTGGAGGCAGAGGT	778	58
	TTTATACAAAAACCTTTTATTGACTG

Note: Primer sequences were designed by Primer 5.0, and according to Wang C, et al. (Wang C et al. 2013) and Rui Z, et al. (Rui et al. 2014).

Figure 1. (A). PCR amplification of exon 15 of BRAF gene. M: DNA marker, 1: negative control, 2: gingiva, 3–7: ameloblastoma specimens; (B). DHPLC chromatograms of exon 15 of BRAF gene with no mutation: negative control; (C). DHPLC chromatograms of exon 15 of BRAF gene with V600E mutation: positive control; (D). DHPLC chromatograms of exon 15 of BRAF gene in normal gingiva; (E). DHPLC chromatograms of exon 15 of BRAF gene mutation in ameloblastoma specimens; (F). Sequencing analysis of exon 15 of BRAF gene in normal gingiva (arrow, GTG); (G). Sequencing analysis of exon 15 of BRAF gene in ameloblastoma specimens (arrow, GAG). PCR, polymerase chain reaction

Figure 2. (A). PCR amplification of exon 3 of SMO gene. M: DNA marker, 1: negative control, 2: gingiva, 3–7: ameloblastoma specimens; (B). PCR amplification of exon 5 of SMO gene. M: DNA marker, 1: negative control, 2: gingiva, 3–7: ameloblastoma specimens; (C). PCR amplification of exon 6 of SMO gene. M: DNA marker, 1: negative control, 2: gingiva, 3–7: ameloblastoma specimens; (D). PCR amplification of exon 10 of SMO gene. M: DNA marker, 1: negative control, 2: gingiva, 3–7: ameloblastoma specimens; (E). PCR-SSCP analysis of exon 3 of SMO gene. M: DNA marker, 1: negative control, gingiva: 2–8: ameloblastoma specimens. The result of lane 3 cannot be interpreted. Abnormal migrational bands (white arrow); (F). PCR-SSCP analysis of exon 5 of SMO gene. M: DNA marker, 1: negative control, gingiva: 2–8: ameloblastoma specimens. Abnormal migrational bands (white arrow); (G). PCR-SSCP analysis of exon 6 of SMO gene. M: DNA marker, 1: negative control, gingiva: 2–8: ameloblastoma specimens. Abnormal migrational bands (white arrow)’; (H). PCR-SSCP analysis of exon 10 of SMO gene. M: DNA marker, 1: negative control, gingiva: 2–8: ameloblastoma specimens. Abnormal migrational bands (white arrow). PCR-SSCP, polymerase chain reaction-single-strand conformation polymorphism; PCR, polymerase chain reaction

Figure 3. (A). Sequencing analysis of exon 2 of SMO gene in wild type ameloblastoma specimens; (B). Sequencing analysis of exon 2 of SMO gene in mutated ameloblastoma specimens (blue arrow, GAA→GAG); (C). Sequencing analysis of exon 3 of SMO gene in wild type ameloblastoma specimens; (D). Sequencing analysis of exon 3 of SMO gene in mutated ameloblastoma specimens (blue arrow, GAA→GAG); (E). Sequencing analysis of exon 5 of SMO gene in wild type ameloblastoma specimens; (F). Sequencing analysis of exon 5 of SMO gene in mutated ameloblastoma specimens (blue arrow, GCG→GCA).

Figure 4. (A). Sequencing analysis of exon 5 of SMO gene in wild type ameloblastoma specimens; (B). Sequencing analysis of exon 5 of SMO gene in mutated ameloblastoma specimens (blue arrow, ACC→AAC); (C). Sequencing analysis of exon 6 of SMO gene in wild type ameloblastoma specimens; (D). Sequencing analysis of exon 6 of SMO gene in mutated ameloblastoma specimens (blue arrow, GGC→GGG); (E). Sequencing analysis of exon 10 of SMO gene in wild type ameloblastoma specimens; (F). Sequencing analysis of exon 10 of SMO gene in mutated ameloblastoma specimens (blue arrow, AGC→ACC).

Table 3. SMO gene mutations in ameloblastoma.

Exon	Nucleotide position	Codon	Amino acid	Mutation type	dbSNP rs cluster id	Case numbers
2	808	GAA→GAG	E176E	Synonymous	rs114834844	1
3	862	GAA→GAG	E194E	Synonymous	rs56334250	9
5	1371	ACC→AAC	T364N	Missense	–	5
	1417	GCG→GCA	A379A	Synonymous	rs35678076	6
6	1444	GGC→GGG	G388G	Synonymous	rs2228617	12
10	2049	AGC→ACC	S590T	Missense	rs114406835	6

Table 4. Relation between BRAF gene mutations and clinical pathology in 30 cases.

Clinical pathology	Class	Case number	Mutation number (V600E)	Mutation rate (%)	P*
Age	y ≤ 20	3	2	66.7
	y > 20	27	16	59.3	1.000
Sex	M	16	11	68.8
	F	14	7	50.0	0.457
Location	Mandible	27	17	63.0
	Maxilla	3	1	33.3	0.548
Histologic type	Follicular	10	7	70.0
	Plexiform	9	6	66.7
	Unicystic	11	5	45.5	0.525
Capsular invasion	Y	5	4	80.0
	N	25	14	56.0	0.622
Recurrence	Y	12	10	83.3
	N	18	8	44.4	0.058

* Fisher’s exact test was used to analyze the relationship between BRAF V600E mutation and clinicopathological features, and a P value < 0.05 was considered significant.

Table 5. Relation between SMO gene mutations and clinical pathology in 30 cases.

Clinical pathology	Class	Case number	Mutation number (T364N, S590T)	Mutation rate (%)	P*
Age	y ≤ 20	3	3	100.0
	y > 20	27	8	29.6	0.041
Sex	M	16	7	43.8
	F	14	4	28.6	0.466
Location	Mandible	27	10	37.0
	Maxilla	3	1	33.3	1.000
Histologic type	Follicular	10	2	20.0
	Plexiform	9	4	44.4
	Unicystic	11	5	45.5	0.454
Capsular invasion	Y	5	4	80.0
	N	25	7	28.0	0.047
Recurrence	Y	11	4	36.7
	N	19	7	36.8	1.000

* Fisher’s exact test was used to analyze the relationship between SMO mutations and clinicopathological features, and a P value < 0.05 was considered significant.

Rui Z, Li-Ying P, Jia-Fei Q, Ying-Ying H, Feng C, Tie-Jun L. 2014. Smoothened gene alterations in keratocystic odontogenic tumors. Head Face Med. 10:36. doi:10.1186/1746-160X-10-36.

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Data availability statement

The data that support the findings of this study are available in the Zenodo repository at https://doi.org/10.5281/zenodo.8267153.