Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder

Table 1 Primer sequences used for polymerase chain reaction - restriction fragment length polymorphism

Gene	Target codon	Strand	Primer sequences
H-Ras	12	+	5’GACGGAATATAAGCTGGTGG 3’
		-	5’AGGCACGTCTCCCCATCAAT 3’
K-Ras	12 and 13	+	5’ACTGAATATAAACTTGTGGTAGTTGGACCT 3’
		-	5’TTCTCCATCAATTACTACTTGCTTCCTGTA 3’
N-Ras	61	+	5’GACATACTGGATACAGCTGGC 3’
		-	5’CCTGTCCTGATGTATTGGTC 3’

+: Forward strand; -: Reverse strand.

Table 2 Summary of Ras gene assays

Gene	Target codon	Restriction enzyme/site	Fragment size
			Undigested	Mutant after digestion	Wild-type/normal after digestion
K-Ras	12 Glycine (GGT) to Valine (GTT)/Aspartic acid (GAT)/Serine (AGT)/Arginine (CGT)/Alanine (GCT)/Cysteine (TGT)	MvaI (BstNI)	144 bp	144 bp	115 bp and 29 bp
		CC↓ WGG
		GGW↑ CC
K-Ras	13 Glycine (GGC) to Aspartic acid (GAC)	HphI	144 bp	101 bp and 43 bp	144 bp
		GGTGAN8↓
		CCACTN7↑
H-Ras	12 Glycine (GGC) to Valine (GTC)/Aspartic acid (GAC)/Serine (AGC)/Alanine (GCC)	Mspl (HpaII)	420 bp	420 bp	390 bp and 30 bp
		(C↓ CGG)
		(GGC↑ C)
N-Ras	61 Glutamine (CAA) to Arginine (CGA)/Lysine (AAA)/Leucine (CTA)	Mlsl (MscI)	65 bp	65bp	44 bp and 21 bp
		(TGG↓ CCA)
		(ACC↑ GGT)

bp: Base pair.

Table 3 Primer sequences used in direct DNA Sequencing

Gene	Strand	Coding exon	Primer sequences	Length of amplified fragment
K-Ras	+	1	F 5'-TTAACCTTATGTGTGACATGTTCTAA-3'	378 bp
K-Ras	-	1	R 5'-CCCTGACATACTCCCAAGGA-3'
K-Ras	+	2	F 5'- TCAAGTCCTTTGCCCATTTT-3'	375 bp
K-Ras	-	2	R 5'- TGCATGGCATTAGCAAAGAC-3'
N-Ras	+	1	F 5'-GCCCAAGGACTGTTGAAAAA-3'	477 bp
N-Ras	-	1	R 5'-TGCATAACTGAATGTATACCCAAAA-3'
N-Ras	+	2	F 5'-GGCAGAAATGGGCTTGAATA-3'	424 bp
N-Ras	-	2	R 5'-CCTAAAACCAACTCTTCCCATAA-3'
H-Ras	+	1	F 5'-GTGGGTTTGCCCTTCAGAT-3'	386 bp
H-Ras	-	1	R 5'-TCTAGAGGAAGCAGGAGACAGG-3'
H-Ras	+	2	F 5'-CAGGACACAGCCAGGATAGG-3'	492 bp
H-Ras	-	2	R 5'-ACATGCGCAGAGAGGACAG-3'

F: Forward strand (+); R: Reverse strand (-); bp: Base pair.

Table 4 Clinicohistopathological profile of patients with urothelial carcinoma of bladder

Clinicohistopathological variables	n (%)
Total no. of patients	87 (100)
Age (yr) mean, range	58.3, 25-83
n < 60	44 (50.17)
n ≥ 60	43 (49.42)
Gender
Male	81 (93.10)
Female	8 (9.19)
Hematuria
Present	87(100)
Absent	Nil
No information	Nil
Smoking/Tobacco chewing status
Smokers	66 (75.86)
Non-smokers	21 (24.1)
Tumor grade
Low	34 (39.04)
High	53 (60.91)
Tumor stage
Ta-T1 (Low/NMIBC)	42 (48.27)
T2-T4 (High/MIBC)	45 (51.72)
Tumor type
Primary	61 (70.11)
Recurrent	26 (29.88)
Tumor Size
> 3 cm	20 (22.98)
< 3 cm	67 (77.01)

NMIBC: Non-muscle invasive bladder cancer; MIBC: Muscle invasive bladder cancer.