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In Silico Analysis of BRCA1 and BRCA2 Sequence Variants of Unknown Clinical Significance
Petar Ozretić
03.12.2007.
• polymorphisms, mutations & unclassified variants (UVs)
• BIC (The Breast Cancer Information Core Database)– BRCA1 : 1643 different sequence variants– BRCA2 : 1856 --- | | ---
• 1/3 BRCA1 & ½ BRCA2 variants from BIC are UVs• difficult to make population based studies
– low frequency alleles– ethnic specificty
• genetic counseling - problem for risk assessment – noninformative test results
• prophylactic mastectomy and/or oophorectomy?!
BRCA1 sequence variants in 220 healthy Croatian women:
Nucleotide change AA change
Proportion of carriers
Allele frequency
Exon BIC accession No. or reference
Times reported in BIC
Clinically important
Missense Variants (11)
3667A>G K1183R 49.09% 27.50% 11 1099 33 No
4956A>G S1613G 46.82% 24.77% 16 1140 36 No
3232A>G E1038G 46.36% 24.55% 11 1087 37 No
2731C>T P871L 43.18% 22.05% 11 1067 26 No
1186A>G Q356R 11.36% 5.68% 11 2558 82 Unknown
3238G>A S1040N 4.09% 2.05% 11 1089 45 Unknown
5075G>A M1652I 2.73% 1.36% 16 1143 39 Unknown
4654G>T S1512I 1.36% 0.68% 15 1136 53 No
2196G>A D693N 0.91% 0.45% 11 1045 16 No
4158A>G R1347G 0.91% 0.45% 11 1351 154 Unknown
2121C>T L668F 0.45% 0.23% 11 2313 25 Unknown
Synonymous Variants (4)
4427T>C S1436S 42.27% 25.23% 13 1128 35 No
2201C>T S694S 44.55% 24.77% 11 1047 14 No
2430T>C L771L 40.00% 20.23% 11 1055 25 No
2030T>C T637T 0.45% 0.23% 11 - Nil
Intronic Variants (6)
IVS17+65G>A 45.91% 26.59% 17 [12]
IVS7-34C>T 43.18% 22.95% 8 1424 9 No
IVS17-53C>T 0.91% 0.45% 18 10455 2 Unknown
IVS20+60ins12 0.91% 0.45% 20 1292 26 Unknown
IVS6+7G>A 0.45% 0.23% 6 2238 12 Unknown
IVS19+49delTA 0.45% 0.23% 19 - Nil
BRCA2 sequence variants in 115 healthy Croatian women:
Nucleotide change
AA change
Proportion of carriers
Alelle frequency
Exon BIC accession No. or reference
Times reported in BIC
Clinically important
Missense Variants (13)
1342A>C N372H 35.65% 19.13% 10 1668 9 No
1093A>C N289H 8.70% 4.35% 10 1129 13 No
3199A>G N991D 8.70% 4.35% 11 1903 6 No
5972T>C M1915T 5.22% 2.61% 11 1108 7 Unknown
1206C>A S326R 3.48% 1.74% 10 1222 105 No
3743C>T S1172L 2.61% 1.30% 11 1908 43 Unknown
7772C>T T2515I 2.61% 1.30% 15 1547 71 No
353A>G Y42C 0.87% 0.43% 3 1004 141 Unknown
3546C>A S1106R 0.87% 0.43% 11 - Nil
4486G>T D1420Y 0.87% 0.43% 11 1274 191 No
5018C>A S1596Y 0.87% 0.43% 11 - Nil
6328C>T R2034C 0.87% 0.43% 11 1324 97 Unknown
7163A>T D2312V 0.87% 0.43% 12 5423 5 Unknown
Synonymous Variants (14)
3624A>G K1132K 46.09% 23.91% 11 1661 8 No
7470A>G S2414S 43.48% 23.04% 14 1125 10 No
4035T>C V1269V 33.04% 19.57% 11 1662 3 No
1593A>G S455S 8.70% 4.35% 10 1106 7 No
2457T>C H743H 7.83% 3.91% 11 13616 7 No
6492T>C T2088T 1.74% 0.87% 11 - Nil
426A>G Q66Q 0.87% 0.43% 3 [13]
687A>C P153P 0.87% 0.43% 5 - Nil
2166C>T S646S 0.87% 0.43% 11 2907 2 No
3744G>A S1172S 0.87% 0.43% 11 3501 4 No
4296G>A L1356L 0.87% 0.43% 11 3525 3 Unknown
5427C>T S1733S 0.87% 0.43% 11 1941 2 No
9315G>A A3029A 0.87% 0.43% 23 - Nil
10092A>G T3288T 0.87% 0.43% 27 - Nil
Intronic and Non Coding Variants (5)
IVS16-14C>T 73.04% 46.52% 17 1126 15 No
IVS11+80del4 61.74% 38.26% 11 2022 1 Unknown
203G>A 55.65% 35.65% 2 1666 12 No
IVS4+67A>C 8.70% 4.35% 4 [14]
IVS24-16T>C 0.87% 0.43% 25 1123 6 Unknown
Truncating Mutations (3)
5873C>A S1882X 0.87% 0.43% 11 1036 26 Yes
10204A>T K3326X 0.87% 0.43% 27 1179 293 No
10323delCins11 3369X 0.87% 0.43% 27 1788 11 No
Deletions (1)
4640delGAA R1471del 0.87% 0.43% 11 - Nil
9 novel BRCA1 & BRCA2 sequence variants by mutation type:
Intronic Variants:BRCA1 IVS19+49delTA
Synonymous Variants:BRCA1 2030T>C (T637T)BRCA2 6492T>C (T2088T)
687A>C (P153P)9315G>A (A3029A)10092A>G (T3288T)
Missense Variants:BRCA2 3546C>A (S1106R)
5018C>A (S1597Y)
Deletions:BRCA2 4640delGAA (R1471del)
• the type of mutation (missense, nonsense, synonymous, frameshift, deletion, insertion...)
• the location of the mutation within the gene, e.g. functional domains, splice sites
• the presence or absence of the variant in a control population
• co-segregation, or lack of co-segregation, of the variant and disease within families
• co-occurrence with a deleterious mutation• the type of amino acid change• the conservation of the amino acid across species• biochemical/functional analysis
Informations needed to assess clinical significance of a variant:
Intronic Splicing
Splice site consensus sequences (from Blencowe, 2000)
• human - 8.8 introns per gene on average, mean intron length 4400 nt, mean exon length 165 nt (5’ donor, 3’ acceptor)
Splicing in outline. Cleavage of the 5′ splice site is promoted by the hydroxyl (OH) attached to the 2′-carbon of an adenosine nucleotide within the intron sequence. This results in the lariat structure and is followed by the 3′-OH group of the upstream exon inducing cleavage of the 3′ splice site. This enables the two exons to be ligated, with the released intron being debranched and degraded. (from Brown, T.A.: Genomes. New York and London: Garland Science; c2002)
Models of SR protein action in exonic-splicing-enhancer-dependent splicing (from Cartegni et al., 2003)
Exonic Splicing Enhancers• ESEs (exonic splicing enhancers) - motifs located in exon regions
– 4-18 nucleotides, degenerative, overlaping, purine-rich– required for regulation of constitutive and alternative splicing (35%-60% of
human genes undergo alternative splicing)– binds splicing regulatory proteins:
• SR-family proteins (serine/arginine-rich)– N-terminal RRMs – RNA recognition motifs – RNA-proteins
interactions– C-terminal RS domain – protein-protein interactions
· ESSs (exonic splicing silencers) - heterogeneous nuclear
ribonucleoproteins (hnRNP A1)
Splicing mutations• up to 50% of all point mutations responsible for genetic diseases
cause aberrant splicing• such mutations can disrupt splicing by directly inactivating or
creating a splice site, by activating a cryptic splice site or by interfering with splicing regulatory elements
• ECEs can be disrupted by single nonsense, missense and translationaly silent point mutations
Two aberrant forms of splicing. (A) In exon skipping the aberrant splicing results in an exon being lost from the mRNA. (B) When a cryptic splice site is selected, part of an exon might be lost from the mRNA, as shown here, or if the cryptic site lies within an intron then a segment of that intron will be retained in the mRNA. (from Brown, T.A.: Genomes. New York and London: Garland Science; c2002)
BDGP: Splice Site Prediction by Neural Network (NNSPLICE)
http://www.fruitfly.org/seq_tools/splice.html
Score probability for the recognition of actual intron donor and acceptor sites
NNSPLICE SpliceSiteFinder donor acceptor donor
acceptorBRCA1 IVS19 w.t. 1.0 0.73 100.0 91.3BRCA1 IVS19+49delTA 1.0 0.73 100.0 91.3
Slovenian founder mutation BRCA2 IVS16-2A>G
SpliceSiteFinder
start donor sequence score BRCA2 IVS16 w.t. 10 AG|GTACTC 68.3BRCA2 IVS16-2A>G 10 AG|GTACTC 68.3
start acceptor sequence score start dist. branch seq. score score of D.+B.BRCA2 IVS16 w.t. 4591 CTTTATTTGTTCAG|G 96.1 4576 -26 ATGAT 76.0 172.1BRCA2 IVS16-2A>G 4611 GTGTGACACTCCAG|G 81.7 4579 -43 ATAAT 75.1 156.8
ESE analysis of novel synonymous sequence variants:
• BRCA1 – 2030T>C (T637T)> + SC35
• BRCA2– 687A>C (P153P) > - SRp40, + SC35, +
SF2/ASF– 6492T>C (T2088T) > - SF2/ASF, + SC35
Poly-Phenhttp://genetics.bwh.harvard.edu/pph/
• PolyPhen (=Polymorphism Phenotyping) predicts possible impact of an amino acid substitution on the structure and function of a human protein, using straightforward physical and comparative considerations.
• position-specific independent count (PSIC) score for each variant – difference ≥1.1 considered to be damaging
• higher PSIC score difference – higher functional impact a particular aminoacid substitution has
• BRCA2 newly found missense variants:PSIC score difference prediction
S1106R (3546C>A) 1.773 possibly damagingS1597Y (5018C>A) 1.815 possibly damaging
Lee PH, Shatkay H.F-SNP: computationally predicted functional SNPs for disease association
studies.Nucleic Acids Res. 36(Database issue):D820-4.