Table 1

Mutation analysis results on the NF1 gene with in silico analysis of the sequence variations found for splice site strength and presence of ESE

Canonical splice sites (AG-GT nucleotides)			Within the consensus splice site sequence
	Mutation	SS score	Exon	Mutation	SS score
Exon
7	889−1 G→C	0.64–<0.40	2	61–12 delT	1–1
7	899−2 A→G	0.64–<0.40	3	288+5 G→C	1–0.83
10b	1527+1 G→C	0.98–<0.40	10c	1642–3 C→G	0.99–0.46
11	1642–2 A→G	<0.40–<0.40	11	1642–3 C→G	<0.40–<0.40
12a	1722 C→A	<0.40–<0.40	17	2857–16 T→A	0.99–0.98
17	2857–2 A→G	0.99–<0.40	18	3113+3 ins A	0.99–<0.40
24	4112–2 A→G	0..99–<0.40	31	5941C→T	0.58–0.77
32	5944–1 G→C	0.78–<0.40	45	7907+5 G→A	0.96–<0.40
Intron
1	1–22 G→C	–
7	899–31 del5	–
11	1642–25 T→C	–
32	6084+8 C→G	0.99–0.98
39	7126+37 G→C	–
42	7395–29 G→A	–
46	8050+20 G→A	–

Exon	Mutation	Amino acid	ESEfinder	RESCUE-ESE	3′SS	5′SS
Same sense
2	168 C→T	S56S	↓SRp55	–	1	0.99
5	703 G→A	L234L	–	–	0.99	0.99
6	846 G→A	Q283Q	↓SF2/ASF	Hexamer	0.55	1
22	3867 C→T	F1289F	↓SRp40 ↓SC35	–	0.99	0.92
28	5172 G→A	L1724F	↓SRp40 SF2/ASF	↓ Hexamer	1	0.98
Missense
1	47 G→C	R16P	↑SC35	–	<0.40	0.91
2	92 A→G	H31R	↓SRp55	–	1	0.99
3	269 T→G	L90R	↓SC35	↓ Hexamer	0.79	1
4a	434 T→C	L145P	↑SRp40 ↑SC35	–	0.81	<0.40
4b	526 G→A	D176N	↑SRp55	–	1	1
4b	528 T→A	D176E	–	Hexamer	1	1
8	1098 A→T	R366S	–	↓ Hexamer	0.68	<0.40
10b	1466 A→G	Y489C	↓SRp40	↓ Hexamer	0.98	0.86
10c	1595 T→C	P533L	↓SRp40 ↑SF2/ASF	↓ Hexamer	0.99	0.90.999
11	1658 A→G	H553R	–	↓ Hexamer × 3	<0.40	0.79
11	1700 T→G	V567G	↓SRp55 ↑SRp40	Hexamer × 2	<0.40	0.79
12b	1885 G→A	G629R	↓SRp55 ↑SF2/ASF	–	<0.40	<0.40
16	2530 C→T	L844F	–	–	0.46	0.92
16	2531 T→C	L844P	–	–	0.46	0.92
21	3610 C→G	R1204	↑SF2/ASF ↑SC35	–	0.9	0.99
Nonsense
3	269 T→G	L90R	↑SRp40,SF2/ASF↓SC35	↓ Hexamer × 2	0.79	1
4b	571 C→T	R192X	↓SRp40	↓ Hexamer	1	1
5	670 del G	V224X	–	–	0.99	0.99
10a	1274 G→A	W425X	↑SRp40	–	0.98	0.99
10a	1318 C→T	R440X	↑SC35	↓ Hexamer × 2	0.98	0.99
13	2041 C→T	R681X	–	–	0.72	0.99
19a	3163 C→T	Q1055X	↓SRp40 SRp55	↓ Hexamer × 2	0.99	<0.40
22	3916 C→T	R1306X	–	Hexamer
23.2	4084 C→T	R1362X	↑SRp40	↓ Hexamer	1	0.98
23.2	4103 T→A	L1368X	↑SRp40	–	1	0.98
27a	4537 C→T	R1513X	↓SF2/ASF	–	0.49	0.53
31	5941C→T	Q1981X	↑SRp55	–	<0.40	<0.40
37	6792 C→G*	Y2264X	–	Hexamer	<0.40	0.99
42	7486 C→T	R2496X	↓SC35	–	0.98	0.73
49	8428 A→T	K2810X	↑SC35	–	1	<0.40

Deletions/insertions
Exon	Mutation	Comments	ESEfinder	RESCUE-ESE	3′SS	5′SS
The table is broken down into the classical mutation categories. Splice site strength (SS score) was evaluated using the web tool found at http://www.fruitfly.org/seq_tools/splice.html and is reported in the table for the wildtype and mutated nucleotides, respectively. Where the splice site strength was below the threshold suggested by the program (<0.40) this is reported as <0.40. The presence of ESE was searched for using both ESEfinder and ESE-RESCUE. The creation or destruction of a site is highlighted by an up or down arrow, respectively. SS, splice site. The consensus is taken as bp within the area analysed by http:www.fruitfly.org/seq_tools/splice.html.
5	12 bp from intron then first 7 bp of exon 5	Destroy 3′ splice site	ND	ND	0.99	0.99
6	787–789 del	253 Val 253 del, in frame deletion	–	Hexamer	0.55	1
7	993 35 bp del	Occurs in exon	↓ 2SRp40 ↓ SC35↓ 2SRp55	↓× 4 Hexamer	0.64	0.93
13	2148 del A	Stop codon created downstream	–	↓× 3 Hexamer	0.72	0.99
16	2508 del A	Stop codon created downstream	–	↓ Hexamer	0.46	0.925
17	Del 2970–2972 AAT	In frame deletion of 991M	–	↓ Hexamer	0.99	1
37	Del 6789 -TTAC	Stop codon created downstream.  >Known not to cause skipping	–	–	<0.40	1
37	6791 ins A	Stop codon created downstream.	–	–	<0.40	1
45	7887 del T	Stop codon created downstream	↑SC35	–	0.52	0.96