Introduction 1 2 BRCA1 BRCA2 3 5 6 7 8 9 BRCA1 BRCA2 BRCA1 BRCA2 10 11 BRCA1 BRCA2 12 BRCA1 BRCA2 BRCA1 BRCA2 13 BRCA1 14 16 BRCA1 BRCA2 BRCA2 17 BRCA1 BRCA2 Materials and methods Patients n n n n DNA extraction and PCR amplification 18 19 17 Denaturing Gradient Gel Electrophoresis and DNA sequencing 17 Multiplex ligation-dependent probe amplification (MLPA) 20 Results and discussions We identified 120 incident Indonesian breast cancer cases diagnosed before the age of 41 years, or having family history of breast cancer, or harboring bilateral breast cancer during September 1999–April 2005 (Jogjakarta) and during July 2004–April 2005 (Jakarta and Denpasar). In addition, 16 of their family members were analyzed. BRCA1 BRCA2 1 2 3 BRCA1 BRCA1 BRCA2 Fig. 1 BRCA2  Fig. 2 A BRCA2 B  Fig. 3 BRCA1  BRCA1 and BRCA2 pathogenic mutations BRCA1 BRCA2 21 BRCA1 BRCA2 n n 1 22 24 Table 1 BRCA1 BRCA2 Patient a gene Exon b mutation type Pathogenic mutation c AE 25 BRCA1 11 c.2784_2785insT frameshift + no B10 31 BRCA1 13 p.Leu1415X nonsense + no AA 40 BRCA1 13–15 d large rearrangement + no AB 34 BRCA2 11 c.3040_3043del4 frameshift + 1 B5 66 BRCA2 11 p.Glu2183X nonsense + no B6 65 BRCA2 11 p.Glu2183X nonsense + no B-III-5 30 BRCA2 11 p.Leu824X nonsense + no AZ 40 BRCA2 11 p.Leu824X nonsense + no W-II 37 BRCA2 21 p.Gln2894X nonsense + no Q-II 40 BRCA1 2 c.101–10T>C IVS ± 6 P-III-19 19 BRCA1 9 p.Val191Ile Missense ± 6 J22 32 BRCA1 11 p.Leu1209Val Missense ? no AZ 40 BRCA1 16 p.Met1652Ile Missense ± 35 B1  24 BRCA1 20 c.5313–31A>G IVS ? no B7 31 BRCA1 24 p.Arg1835Gln Missense ? no 216 33 BRCA1 24 p.Thr1852Ile Missense ? no P-III-19 19 BRCA2 5 p.Gln147Arg Missense ± 6 B3 24 BRCA2 10 p.Gln609Glu Missense ? no C-II-7 39 BRCA2 11 p.Met1149Val Missense ± 5 AO 28 BRCA2 11 p.Met1149Val Missense ± 5 AQ 44 BRCA2 11 p.Met1149Val Missense ± 5 BH 38 BRCA2 11 p.Met1149Val Missense ± 5 172 36 BRCA2 11 p.Gln699Leu Missense ? no J32 29 BRCA2 11 p.Arg2108Cys Missense ± 16 J6 33 BRCA2 11 p.Val950Ile Missense ? no 206 37 BRCA2 25 c.9485–16T>C IVS ± 4 BC 35 BRCA2 27 p.Ile3412Val Missense ± 109 166 33 BRCA2 27 p.Ile3412Val Missense ± 109 J24 35 BRCA2 27 p.Ile3412Val Missense ± 109 206 37 BRCA2 27 p.Lys3326X nonsense  ± 289 a b BRCA1 BRCA2 c d BRCA1 BRCA2 BRCA1 BRCA2 BRCA1 BRCA2 2 BRCA1 BRCA1 25 BRCA1 Table 2 BRCA1 BRCA2 Patient a Gene with germline mutation b stage Diagnosis Menopausal status family history of cancer Survival status AE 25 BRCA1 c.2784_2785insT IIIB/IIIA IDC, bilateral pre No DOD 9 w B10 31 BRCA1 p.Leu1415X I IDC pre No DOD 57 w AA 40 BRCA1 c IIIB IDC N+ pre No AWD AB 34 BRCA2 c.3040_3043del4 IIIB IDC N+ pre Sister, Int DOD 17 w B5 63 BRCA2 p.Glu2183X IV Tubular post Sister,Br AWD B6 65 BRCA2 p.Glu2183X III IDC post Brother, Br AWD B-III-5 30 BRCA2 p.Leu824X I IDC pre No AWD AZ 40 BRCA2 p.Leu824X IV IDC pre Sister, Cv DOD 46 w W-II 37 BRCA2 p.Gln2894X IIIA IDC pre No DOD 107 w a b BRCA1 BRCA2 c IDC: invasive ductal carcinoma; DOD: dead of disease; bil: bilateral breast cancer; N+: with metastatic to lymph node; Int: intestinum cancer, Br: breast cancer; Cv: cervical cancer BRCA2 26 BRCA2 17 1 BRCA2 BRCA1 BRCA2 27 BRCA1 BRCA1 BRCA2 BRCA1 BRCA1 and BRCA2 unclassified variants BRCA1 BRCA2 1 BRCA1 BRCA1 BRCA1 28 BRCA2 1 BRCA2 BRCA2 Mus musculus, Rattus rattus, Bos taurus, Gallus gallus, Canis familiaris, Macaca mullata Monodelphis domestica. Bos taurus BRCA1 Glycosylation moiety of an amino acid also plays a role in protein function. Amino acid substitutions involving Serine, Threonine and Asparagine, should also be checked for their O-GlcNac potential and threshold. Here we have a Threonine to Isoleucine substitution (p.Thr1852Ile) that after checking with YinOYang (http://www.cbs.dtu.dk/services/YinOYang) showed no significant threshold changes between the wildtype and the mutant allele. 29 3 Table 3 BRCA1 BRCA2 Gene Amino acid change Change of charge Change of amino acid group a # species with conserved sequence BRCA1 Leu to Val None No 32 a,b,c,d,e,f,g BRCA1 Arg to Gln Pos to no charge Yes 43 a,c,f,g BRCA1 Thr to Ile polar to non polar Yes 89 a,c,g BRCA2 Gln to Glu No charge to neg Yes 29 a,b,c,g BRCA2 Gln to Leu Polar to non polar Yes 113 a,b,d,e,f BRCA2 Val to Ile None No 29 f,g a 29 a = Macaca mullata, b = Bos taurus, c = Canis familiaris, d = Rattus rattus, e = Mus musculus, f = Gallus gallus, h = Monodelphis domestica Overall, we propose that among the seven novel UVs, there are three mutations that are possibly pathogenic: p.Leu1209Val for its location in a conserved region, and p.Gln609Glu and the p.Gln699Leu because of two adjacent acidic amino acid being formed and a high similarity score, respectively. BRCA1/2 P BRCA1 BRCA2 30 BRCA1 BRCA1 BRCA2. BRCA1 BRCA2