Chapter 3: Results 91 3.1. Genetic alterations of the BRCA1 gene in Singapore Malay women with breast/ ovarian cancer. 3.1.1. BRCA1 mutations The mutation analysis of the entire coding region of BRCA1 performed on blood samples from 76 and 20 Malay breast and ovarian cancer patients respectively, selected for either early-onset or with family history of breast and/or ovarian cancer, revealed twelve different BRCA1 alterations (Tables and 8). These sequence alterations were identified in 16 unrelated patients and they span the entire coding region of BRCA1. They comprised one frameshift mutation (c.2845insA), two missense mutations (c.335C>G and c.778C>A) and nine polymorphisms (c.233G>A, c.432-44G>A, c.420+26CA>AC, c.561-34T>C, c.5106-68A>G, c.5106-92A>G, c.5272+72G>C, c.5272+73G>A and c.5451+85delC). Four of these variants are novel and they are c.778C>A, c.432-44G>A, c.420+26CA>AC and c.5451+85delC (Figure 13). All sequence variants were designated according to the Human Genome Organization (HUGO) recommendations (Den Dunnen JT and Antonarakis SE, 2000). 92 93 81 15 96 (3.8%) (3.3%) (3.6%) NK (2.4%) (11.8%) (3.3%) NK (5.4%) (17.6%) 12 (6.5%) (11.1%) (9.4%) (15.4%) (10.0%) (12.5%) 15 (18.5%) 15 (100.0%) 30 (31.3%) With family history Br/Ov Other Ca 23 (100%) 154 (92.2%) 12 (70.6%) 166 (90.2%) NK 21 (80.8%) 26 (86.7%) 47 (83.9%) 57 (70.4%) 57 (59.4%) With no family history relative. Percentages are in brackets. Br - breast; Ov - ovarian; Ca - cancer; yrs - years; NK - not known Note: Early-onset - 45 years and below for breast cancer and 50 years and below for ovarian cancer. Late-onset - Above 45 years for breast cancer and above 50 years for ovarian cancer. Family history is defined as the presence of cancer on either maternal or paternal side up to second degree Mutation analysis of the recurrent c.2845insA mutation in Singapore Malay women Blood from FTA 26 Early-onset (breast/ovarian cancer) 30 Late-onset (breast/ovarian cancer) 56 All (breast/ovarian cancer) Paraffin-embedded tissues 84 Breast cancer (age of onset unknown) Healthy controls, Malays 167 50 years and below 17 above 50 yrs 184 All Methylation analysis of BRCA1 promoter in Singapore women with breast cancer 23 Matched tumour and normal tissue from same patient Mutation analyses of the entire coding of BRCA1 in Singapore Malay women with breast/ovarian cancer Blood Early-onset Late-onset All Fallopian tube cancer n= Table 7. Summary of BRCA1 genetic / epigenetic study samples. The classification is based on age of onset and family history. 94 Paraffin 11 11 6 17 17 18 18 21 10 11 Exon A878A P871T No change Intronic Intronic Intronic Intronic Intronic Intronic Intronic Intronic S72R S220Y N909frameshiftX5 Amino acid change n=84 n=84 1 2 2 1 2 2 1 n=76 n=96 1 No. of BC cases n=189 Total no. of BC and OC cases n=236 Nil Nil 0 0 0 0 0 n=20 No. of OC cases n=47 Nil 17 Nil Nil Nil Nil Nil # times recorded in BIC* unknown unknown unknown unknown no unknown unknown no no no unknown unknown yes Clinically important** Note: BC - breast cancer, OC - ovarian cancer and BIC - Breast Information Core Database. * Figures accurate as of 20 May 2007. ** Based on the opinion of BIC steering committee that the sequence change interferes with gene function and results in increased risk of cancer (BIC database). Polymorphism c.2754A>C (Novel) Missense mutation c.2731C>A c.5451+85delC (Novel) c.561-34T>C c.5106-68A>G c.5106-92A>G c.5272+72G>C c.5272+73G>A Polymorphism c.233G>A c.432-44G>A (Novel) c.420+26CA>AC(Novel) Blood Blood + Paraffin + FTA cards Frameshift mutation c.2845insA Missense mutation c.335C>G c.778C>A (Novel) DNA source Sequence alteration Table 8. BRCA1 mutations found in Singapore Malay women with breast/ovarian cancer. Blood collected, using FTA cards, from 56 sporadic Malay breast/ovarian patients was investigated for the recurrent BRCA1 c.2845insA mutation only and none of these patients had the recurrent mutation. In addition, 84 paraffin-embedded sporadic Malay breast tumour samples were analyzed for the recurrent mutation and all of them tested negative (Table 8). However, two other novel sequence variants, a missense mutation (c.2731C>A) and a neutral polymorphism (c.2754A>C), were identified. In summary, the recurrent BRCA1 c.2845insA mutation was detected in out of a total of 236 breast and ovarian cancer cases (whole blood, FTA and paraffin sources) (Table 8). This recurrent mutation was found mainly in cases with early-onset of cancer but not in late-onset cases. None of the 184 unrelated age-matched normal subjects was found to carry the recurrent BRCA1 c.2845insA mutation. 3.1.2. BRCA1 mutations, age of onset of cancer and family history of cancer In this study, the recurrent BRCA1 c.2845insA mutation was detected in six unrelated patients of which three had breast cancer and the remaining three had ovarian cancer (Table 8, Figure 12). Out of the six patients with the recurrent BRCA1 c.2845insA mutation, four patients (N536, N587, N610 and N521) had at least a first or second degree relative with breast or ovarian cancer (Table 9). Patients N536, N587 and N610 were early-onset breast cancer cases while N521 was an early-onset ovarian cancer case. The recurrent mutation was also detected in an early-onset ovarian cancer patient, N590, who had a second-degree relative diagnosed with stomach cancer. 95 N Normal (220kDa) a Truncated protein (~106kDa) N b Variant band Variant band Figure 12. The recurrent BRCA1 c.2845insA deleterious mutation identified in six Malay breast and ovarian cancer patients. a. PTT gel and b. SSCP gel. N – normal (healthy control). Breast and ovarian cancer patients are labeled as samples to representing N521, N536, N576, N587, N590 and N610/#014 respectively. The three missense mutations detected in this study were of unknown clinical significance and were found in three separate breast cancer patients. The first missense mutation, c.778C>A (S220Y), was identified in a patient (N536) diagnosed with earlyonset breast cancer whose sister was diagnosed with breast cancer at 33 years of age and father diagnosed with colon cancer at the age of 58 years (Table 9, Figure 13). In addition, this patient was also found to be carrying the deleterious c.2845insA mutation. This missense mutation is novel which causes an amino acid change within the BRCA1 NLS domain (Wu LC, et al, 1996; Bork P, et al, 1997; Hashizume R, et al, 2001). The second missense, c.335C>A (S72R), found in a patient diagnosed with early-onset breast cancer with no family history of cancer, is a rare mutation having only been described once in the BIC database (Table 10, Figure 13). This mutation causes an amino acid 96 change within the BRCA1 RING finger structural domain, which is in close proximity with the RING finger motif. The third missense mutation, c.2731C>A (P871T) identified in a breast cancer patient, T27, of unknown age of diagnosis and family history status has been reported once in the BIC database (Table 10). This mutation occurs within BRCA1 NLS region (Bork P, et al, 1997). Table 9. BRCA1 mutations in Malay patients with family history of cancer. Patient # Breast cancer DNA source from blood N536 Age Family history Mutation summary 35 1. Sister, Breast 33yrs; 2. Father, Colon 58yrs 1. c.2845insA; 914STOPTGA (FS) 2. c.778C>A; Ser220Tyr; S220Y (MM) N587 44 1. Mother, Breast 60yrs 2. Maternal aunt, Breast 50+yrs 1. c.2845insA; 914STOPTGA (FS) 2. c.5272+72G>C; IVS18+72G>C (IP) N610/#014 35 1. Mother, Ovary 49yrs 2. Maternal aunt, cancer type unknown 1. c.2845insA; 914STOPTGA (FS) 2. c.5451+85delC; IVS21+85delC (IP) N599 29 1. Maternal grandfather, Colon 70yrs 1. c.5106-68A>G; IVS16-68A>G (IP) 2. c.5106-92A>G; IVS16-92A>G (IP) 49 1. Sister, Breast 37yrs 2. Half maternal aunt, Breast 30yrs 3. Maternal cousin, Breast 40yrs 1. c.2845insA; 914STOPTGA (FS) N590 40 1. Maternal aunt, Stomach (age unknown) 1. c.2845insA; 914STOPTGA (FS) N658 53 1. Sister, Ovary (age unknown) Ovarian cancer DNA source from blood N521 1. c.5272+73G>A; IVS18+73G>A (IP) Note: yrs - years ; ins - insertion; del - deletion; IVS - intronic variant sequence; IP intronic polymorphism; MM - missense mutation; FS - frameshift mutation. Patients with the framshift c.2845insA mutation are in bold. Out of ten polymorphisms detected in this study, two were neutral polymorphisms and eight were intronic mutations (Table 8, Figure 13). The first neutral polymorphism, c.233G>A (K38K) was detected in two breast cancer patients (N33 and N522) and one 97 ovarian cancer patient (N625). All three carriers were early-onset cases with no family history of cancer. This polymorphism has been described 17 times in the BIC database. The second neutral polymorphism, c.2754A>C (A878A), is a novel mutation and occurs within BRCA1 NLS region. This polymorphism was identified in a breast cancer patient (T36) of unknown age of diagnosis and family history status (Table 10). The eight intronic variants, all detected in breast cancer patients, were IVS5-44G>A, IVS6+26CA>AC, IVS7-34T>C, IVS16-68A>G, IVS16-92A>G, IVS18+72G>C, IVS18+73G>A and IVS21+85delC (Table 8, Figure 12). Four intronic variants, IVS734T>C, IVS16-68A>G, IVS16-92A>G and IVS18+73G>A, have been described previously while the remaining four intronic variants, IVS5-44G>A, IVS6+26CA>AC, IVS18+72G>C and IVS21+85delC, are novel (BIC database). The intronic variants, IVS16-68A>G and IVS16-92A>G, were identified in two unrelated early-onset breast cancer patients, N599 and N616. Patient N599 had a maternal grandfather diagnosed with colon cancer at the age of 70 years while N616 had no family history of cancer (Tables and 10). The BRCA1 variant, IVS7-34T>C, was found in an early-onset breast cancer patient, N511, who had no family history of cancer (Table 10). The intronic variant, IVS18+73G>A, was identified in two samples, a breast and an ovarian cancer patients. Patient N640 was diagnosed with early-onset breast cancer and no family history of cancer while N658, diagnosed with late-onset ovarian cancer, had a stepsister diagnosed with ovarian cancer at an unknown age (Tables and 10). 98 Two of the novel intronic variants, IVS5-44G>A and IVS6+26CA>AC, were identified in the same patient, N591, diagnosed with early-onset breast cancer with no family history of cancer (Table 10). In addition, this patient was also found to be carrying the missense mutation, c.335C>G. The third novel intronic mutation, IVS18+72G>C, was found in an early-onset breast cancer patient whose mother and maternal aunt were diagnosed with breast cancer at 60 and 50+ years of age (Table 9). Meanwhile, the fourth novel intronic variant, IVS21+85delC, was identified in two unrelated early-onset breast cancer patients, N537 and N610. Patient N537 had no family history of cancer while the mother of patient N610 was diagnosed with ovarian cancer at 49 years of age (Tables and 10). In addition, N610 also had a maternal aunt diagnosed with an unknown cancer type and was found to be carrying the deleterious c.2845insA mutation. 99 Table 10. BRCA1 mutations in Malay patients without or unknown family history of cancer. Patient # Breast cancer DNA source from blood N33/N389 Age Family history Mutation summary 38 Nil 1. c.233G>A; Lys38Lys; K38K (NP) N522 32 Nil 1. c.233G>A; Lys38Lys; K38K (NP) N591/A0062 41 Nil 1. c.335C>G; Ser72Arg; S72R (MM) 2. c.432-44G>A; IVS5-44G>A (IP) 3. c.420+26CA>AC; IVS6+26CA>AC (IP) N511 34 Nil 1. c.561-34T>C; IVS7-34T>C (IP) N616 35 Nil 1. c.5106-68A>G; IVS16-68A>G (IP) 2. c.5106-92A>G; IVS16-92A>G (IP) N640 45 Nil 1. c,5272+73G>A; IVS18+73G>A (IP) N537 43 Nil 1. c.5451+85delC; IVS21+85delC (IP) DNA source from paraffin U T27 Unknown 1. c.2731C>A; Pro871Thr; P871T (MM) U Unknown 1. c.2754A>C; Ala878Ala; A878A (NP) T36 Ovarian cancer DNA source from lymphocytes 50 Nil N576 N625 42 Nil 1. c.2845insA; 914STOPTGA (FS) 1. c.233G>A; Lys38Lys; K38K (NP) Note: yrs - years ; ins - insertion; del - deletion; IVS - intronic variant sequence; NP neutral polymorphism; IP -intronic polymorphism; MM - missense mutation; FS frameshift mutation. 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(BRCA1 Intron 20 ) in Singapore Malay women with breast/ovarian cancer 3 .2. 2 Identification of a common haplotype at 3 BRCA1 intragenic markers in breast/ovarian cancer patients with the recurrent BRCA1 c .28 45insA mutation The haplotypes of six breast and ovarian cancer patients with the recurrent BRCA1 c .28 45insA mutation were examined at three BRCA1 intragenic markers All six mutation carriers shared... 50 yrs NA - not applicable Control All (early- and late-onset) Late-onset Early-onset Breast and ovarian cancer Table 15 Common haplotype (159- 127 -1 42 for D17S1 323 -D17S1 322 -D17S855 respectively) in Singapore Malay breast/ovarian women 3 .2. 3 The identification of the recurrent mutation-associated haplotype in a Malay fallopian tube cancer patient Haplotype analysis of all six members of this family with... haplotypes are possibly at an increased risk in developing breast/ovarian cancer with relative risks of 2. 24 and 2. 32 for both haplotypes respectively, with p < 0.01 (Table 17) Table 17 Identification of other haplotypes in Malay women with breast/ovarian cancer Haplotype, D17S1 323 D17S1 322 D17S855 Early-onset breast/ovarian cancer, n=84 Late-onset breast/ovarian cancer, n=40 All breast/ovarian cancer,... as shown above, with reduced peaks for the corresponding exons (denoted by arrows) was used as positive control for BRCA1 genomic rearrangement analysis in Malay samples (Lim YK, et al, 20 07) 104 3 .2 BRCA1 haplotype analysis 3 .2. 1 Haplotyping of Malay breast/ovarian cancer patients and the general Malay population Three BRCA1 intragenic markers, D17S1 323 (Intron 12) , D17S1 322 (Intron 19) and D17S855... 19) marker in Singapore Malay women with breast/ovarian cancer 45 40 35 25 20 15 10 5 12 627 12 425 12 223 12 021 11 819 11 617 11 415 11 21 3 11 011 10 89 0 10 67 % of alleles 30 Allele size, bp Early-onset breast/ovarian cancer, n=78 Late-onset breast/ovarian cancer, n=40 All breast/ovarian cancer, n=118 Control, n=164 109 110 0.9 7.6 25 0.09 12. 1 30 A c.335C>G IVS21+85delC d a Exon 10 1 2 Exon 18 1 2 c.778C >A IVS18+73G >A b e Exon 8 1 2 Exon 17 1 2 IVS7-34T>C c IVS16-6 8A> G and IVS16-9 2A> G f IVS5-44G >A and IVS6 +26 CA>AC g Figure 13 Ten BRCA1 genetic variants detected in Malay women with breast/ovarian cancer by SSCP a – Exon 3 c .23 3G >A (patient N33/389); b – Exon 10 c.778C >A (patient N536); c... cancer patients A N4 52 - with allele sizes of 143 and 151 bases at D17S1 323 (Intron 12) ; B N 523 - with allele sizes of 116 and 122 bases at D17S1 322 ; and C N601 - with allele sizes of 157 and 163 bases at D17S855 Arrows in bold indicate allele peaks B C 106 Figure 18 Allele distribution of the D17S1 323 (BRCA1 Intron 20 ) marker in Singapore Malay women with breast/ovarian cancer 45 40 35 25 20 15 10 5 13... Malay breast and ovarian cancer patients analyzed A novel BRCA1 exon 13-15 deletion, identified in an ovarian cancer patient of Indian ethnicity, was used as a positive control in the rearrangement screening (Figure 16) This was part of a study carried out in 103 our laboratory in which three different BRCA rearrangements were detected in three unrelated non-Malay Singapore patients with breast/ovarian . (breast/ovarian cancer) Late-onset (breast/ovarian cancer) Mutation analysis of the recurrent c .28 45insA mutation in Singapore Malay women Blood from FTA All (breast/ovarian cancer) Late-onset All . cancer 0 5 10 15 20 25 30 35 40 45 10 6 -7 108-9 110-11 1 1 2 -1 3 114-15 1 1 6 -1 7 1 1 8 -1 9 1 2 0 -2 1 122 -23 124 -25 1 2 6 -2 7 Allele size, bp % of alleles Early-onset breast/ovarian cancer, n=78 Late-onset breast/ovarian cancer, n=40 All breast/ovarian cancer, n=118 Control,. were early-onset breast cancer cases while N 521 was an early-onset ovarian cancer case. The recurrent mutation was also detected in an early-onset ovarian cancer patient, N590, who had a second-degree