Table 1. Validation of the mini-gene splicing assay investigating 24 BRCA1 variants previously examined by RT-PCR analysis on RNA from blood samples or LCLs.

NT change (HGVS)	NT change (BIC)	Location	Effect observed in mini-gene assay	Effect observed in RT-PCR analysis on RNA from blood samples or LCLs	RNA change (HGVS)	References	5-Tier splicing classificationa
c.-19−10T>C	101−10T>C	Intron 1	No aberration	No aberration	r.[=]	15	Class 2
c.81−14C>T	200−14C>T	Intron 2	No aberration	No aberration	r.[=]	16, 28	Class 2
c.134+3A>C	253+3A>C	Intron 3	In-frame skipping of exon 3	In-frame skipping of exon 3	r.[81_134del]	16	Class 5
c.212+3A>G	331+3A>G	Intron 5	Increased in-frame skipping of exon 5 and out-of-frame skipping of the last 22 nucleotides of exon 5	In-frame skipping of exon 5 and out-of-frame skipping of the last 22 nucleotides of exon 5	r.[135_212del, 191_212del]	16, 18, 28	Class 5
c.301+7G>A	420+7G>A	Intron 6	No aberration	No aberration	r.[=]	28	Class 2
c.441+1G>A	560+1G>A	Intron 7	Skipping of exon 7 and out-of-frame deletion of 62 bp of exon 7	Out-of-frame deletion of 62 bp of exon 7	r.[302_441del, 380_441del]	18	Class 5
c.547+2T>A	666+2T>A	Intron 8	Skipping of exon 8	Skipping of exon 8	r.[442_547del]	27, 28	Class 5
c.593+8A>G	712+8A>G	Intron 9	No aberration	No aberration	r.[=]	18	Class 2
c.594−2A>C	713−2A>C	Intron 9	Skipping of exon 10b and in-frame retention of 21 bp of intron 9	Skipping of exon 10 and in-frame retention of 21 bp of intron 9 (as well as other deletions identified with primers spanning more exons)	r.[594_670del, 593_594ins 594-21_594-1; 594-2a>c]	24, 29	Class 3
c.670+8C>T	789+8C>T	Intron 10	Increased inclusion of exon 10	Increased inclusion of exon 10 (and 9)	r.[=]	11	Class 3
c.4357+1G>A	4476+1G>A	Intron 13	Skipping of exon 13	Skipping of exon 13	r.[4186_4357del]	9	Class 5
c.4484+1G>A	4603+1G>A	Intron 14	Skipping of exon 14	Skipping of exon 14	r.[4358_4484del]	20, 28	Class 5
c.4484+14A>G	4603+14A>G	Intron 14	No aberration	No aberration	r.[=]	28	Class 2
c.4675+1G>A	4794+1G>A	Intron 15	Skipping of exon 15	Skipping of exon 15 (as well as other smaller deletions)	r.[4485_4675del]	26	Class 4
c.4986+6T>G	5105+6T>G	Intron 16	Out-of-frame retention of 65 bp of intron 16 and increased skipping of exon 16	Out-of-frame retention of 65 bp of intron 16	r.[4986_4987ins4986+1_4986+65; 4986+6u>g, 4676_4986del]	18	Class 5
c.5074+1G>T	5193+1G>T	Intron 17	Retention of 153 bp of intron 17 and skipping of exon 17	Retention of 153 bp of intron 17	r.[5074_5075ins5074+1_5075+153; 5074+1g>u, 4987_5074del]	22	Class 5
c.5074+6C>G	5193+6C>G	Intron 17	Minor increased skipping of exon 17	No aberration (data not shown)	r.[4987_5074del]	21, 28	Class 3
c.5153−1G>A	5272-1G>A	Intron 18	Out-of-frame deletion of 1 bp of exon 19	Out-of-frame deletion of 1 bp of exon 19	r.[5153del]	23	Class 5
c.5277+1G>A	5396+1G>A	Intron 20	In-frame retention of 87 bp of intron 20 and skipping of exon 20	In-frame retention of 87 bp of intron 20 and skipping of exon 20	r.[5277_5278ins5277+1_5277+87; 5277+1g>a, 5194_5277del]	24	Class 5
c.5332+1G>A	5451+1G>A	Intron 21	Skipping of exon 21	Skipping of exon 21	r.[5278_5332del]	27	Class 5
c.5406+1G>A	5525+1G>A	Intron 22	Skipping of exon 22	Skipping of exon 22	r.[5333_5406del]	28	Class 5
c.5406+8T>C	5525+8T>C	Intron 22	No aberration	No aberration	r.[=]	11, 28	Class 2
c.5406+33A>T	5525+33A>T	Intron 22	No aberration	No aberration	r.[=]	28	Class 2
c.5467+1G>A	5586+1G>A	Intron 23	Skipping of exon 23	Skipping of exon 23	r.[5407_5467del]	25	Class 5

Abbreviation: LCL, lymphoblastoid cell line.

^aThe 5-Tier splicing classification is based on Whiley et al.²⁹ and Spurdle et al.³⁰

^bAlso observed in wild-type control.