Large genomic rearrangements of the BRCA1 and BRCA2 genes ...

Breast Cancer Res Treat DOI 10.1007/s10549-010-0817-z

PRECLINICAL STUDY

Large genomic rearrangements of the BRCA1 and BRCA2 genes: review of the literature and report of a novel BRCA1 mutation Michelle D. Sluiter • Elizabeth J. van Rensburg

Received: 26 January 2010 / Accepted: 22 February 2010 Ó Springer Science+Business Media, LLC. 2010

Abstract Germline mutations in BRCA1 and BRCA2 increase the risk for developing breast and ovarian cancer. Previously, the techniques available allowed only for the identification of small genomic alterations, but the dawn of new technology now allows for the rapid detection of large genomic rearrangements (LGRs). LGRs in BRCA1 are responsible for between 0 and 27% of all BRCA1 diseasecausing mutations identified in numerous populations. Such alterations are far less common in the BRCA2 gene. To determine the impact of BRCA1 and BRCA2 LGRs in South Africa, 52 hereditary breast and/or ovarian South African families (36 were Afrikaners) were screened for BRCA1 and BRCA2 LGRs using multiplex ligationdependent probe amplification. These patients were previously shown to be BRCA1 and BRCA2 small mutation negative. One LGR was detected in BRCA1 in a South African family with Greek ancestry. This is a novel deletion of both exons 23 and 24 (NG_005905.2:g.169527_ 180579del). This first study of BRCA rearrangements in South Africa reveals that LGRs comprise *3% of identified BRCA1 mutations, a low rate in comparison to other populations. In addition, we have reviewed all 98 previously characterized BRCA1/2 LGRs and re-named them according to the recommended HGVS nomenclature, using the recently released RefSeqGene records, NG_005905.2 and NG_012772.1 for BRCA1 and BRCA2. A standardized resource is now provided which will assist researchers in determining whether their LGRs are novel. Furthermore, we have clarified some of the previously misunderstood

M. D. Sluiter (&) E. J. van Rensburg Department of Genetics, University of Pretoria, P.O Box 2034, Pretoria 0001, South Africa e-mail: [email protected]

rules of nomenclature, which will make uniform reporting of BRCA1/2 easier in the future. Keywords BRCA1 BRCA2 Large genomic rearrangements Breast cancer Uniform nomenclature

Introduction Breast cancer is the second most common form of cancer to afflict women in South Africa, and overall the lifetime risk for developing cancer of the breast is 1 in 31 for South African women, ranging from 1 in 13 for white women to 1 in 57 for black women [1]. Hereditary breast cancer is responsible for 5–9% of all breast cancer cases [2], and inheritance of a germline mutation within one of the two breast cancer susceptibility genes, BRCA1 and BRCA2, confers a high cumulative risk of breast and/or ovarian cancer [3, 4]. Mutation screening of these two genes has, in the past, fallen short in terms of identification of diseasecausing mutations [5], i.e., not all ‘‘familial cases’’ are explained by these two genes. This could either mean that other breast cancer susceptibility genes exist, or alternatively that a large number of causative mutations were missed due to the use of classical qualitative PCR-based techniques that are incapable of detecting large genomic rearrangements (LGRs). BRCA1 is rich (41.5%) in Alu sequences [6], which are known to mediate the occurrence of rearrangements. It is therefore not surprising that at least 81 different LGRs have been characterized in BRCA1. BRCA2 contains fewer Alu repeats, which may, to some extent, explain why fewer rearrangements have been reported in this gene [7–15]. A large number of techniques have now become available for the detection of large deletions and duplications. Currently, multiplex

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ligation-dependent probe amplification (MLPA) is the mostcommonly used technique for the detection of such mutations in the BRCA genes. The first LGR in BRCA1 (1-kb deletion of exon 17) was detected in 1997 by Southern blotting [16]. Following this report, Swensen et al. [17] found a 14-kb deletion, which removes exons 1a, 1b, and 2 of BRCA1. Many other groups subsequently employed quantitative techniques, and it is now clear that, as with small mutations, the contribution of LGRs is variable between different populations, and numerous founder rearrangements have been identified. The contribution of these LGRs varies from 0 to 27% of BRCA1 mutation positive families in Iranian/French Canadian and Dutch populations, respectively [18–20]. Perhaps, the most striking founder mutations identified so far are the 510-bp deletion of exon 22, the 3.8-kb deletion of exon 13 (both found in the Dutch population), and the 6-kb insertion of exon 13 that is common amongst Europeans where the ancestor is possibly of northern British origin [21]. In some populations, LGRs are so frequent that it has been suggested that MLPA will be more costeffective if used as an initial phase screen [8, 22, 23]. Previous studies on the Afrikaner population have indicated that small mutations in BRCA1 and BRCA2 account for 19 and 46.5% of familial breast/ovarian cancer susceptibility, respectively, where founder effects were observed in both the genes [24]. The contribution of these genes to the burden of breast cancer in South Africa may however be an underestimation, due to the fact that quantitative techniques have not been used previously. In an attempt to determine the impact of BRCA1 and BRCA2 large genomic alterations on the burden of the disease in the South African population, 52 high-risk families were screened by MLPA for both BRCA1 and BRCA2.

Materials and methods Patient recruitment Sixty-six South African patients from 52 breast/ovarian cancer families, previously shown to be negative for small BRCA1 and BRCA2 mutations by means of SSCP/Hetroduplex Analysis and PTT, were included in this study. Patients were referred by private practitioners, or attended the Familial Cancer Clinic of the Department of Human Genetics at the University of Pretoria. Written informed consent was obtained from each patient for participation in the study that was approved by the Ethics Committee of the faculty of Health Sciences, University of Pretoria. The selection criteria of the families included in the study was based on the occurrence of three or more breast and/or

123

ovarian cancer cases within a single family, where bilateral disease was scored as two cases. However, two families with only two breast cancer cases (consisting of one female and one male breast cancer case each) were included in the study. These two families were included because of the association between male breast cancer and BRCA2 mutations. In total, 36 Afrikaner and 3 Ashkenazi Jewish families were screened, together with South African families of English (7), Dutch (2), Greek (1), Polish (1), Portuguese (1), and German (1) ancestry. MLPA and data analysis Genomic DNA was extracted from EDTA blood as previously described by Johns and Paulus-Thomas [25]. MLPA [26] was performed using the P002 and P087 kits for BRCA1 and the P045 kit for BRCA2 (MRC-Holland, Amsterdam, the Netherlands), according to the manufacturer’s instructions. Amplified products were separated using a ABI-3130 (Applied Biosystems, Foster City, CA, USA) genetic analyzer. MLPA data were analyzed using the National Genetics Reference Laboratories (NGRL), Manchester analysis sheet (www.ngrl.org.uk/Manchester) or by calculating relative peak areas (RPAs) for each sample as recommended by the manufacturer (www.mrcholland.com). Characterization of detected deletions In order to characterize the breakpoints of suspected LGRs, a series of long-range PCR formats were designed to successfully narrow down the breakpoint regions. Long-range PCR was performed using LA Taq (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s recommendations. The primer pair that eventually allowed for characterization of the exon 23–24 deletion breakpoints was BRC1-23BF: 50 -ctc ccc ctc ctc tct gtg act-30 /BRC1-3’ER 50 -ctg gtc ctg gag gag gag tt-30 . In order to sequence the deletion fragments, 45-ll PCR product was electrophoresed on ethidium bromide stained 0.76% FMC SeaPlaqueÒGTGÒ agarose gel. The deletion fragment was excised from the gel and the agarose removed using the WizardÒ SV Gel and PCR Clean-Up System (Promega), using the centrifugation protocol without modification. Cycle sequencing was performed using the BigDyeÒ Terminator v3.1 Cycle Sequencing kit and sequence analysis was performed on the ABI 3130 (Applied Biosystems, Foster City, CA, USA). Genomic breakpoints were designated according to the HGVS nomenclature criteria [27] using the BRCA1 reference sequence, NG_005905.2, obtained from the NCBI RefSeq database (http://www. ncbi.nlm.nih.gov/RefSeq/RSG/).


Results Fifty-two unrelated families were screened for LGRs at the two breast cancer gene loci. Two of these families showed evidence consistent with deletions of one or more exons within BRCA1. No indication of a BRCA2 rearrangement was obtained. Reductions in dosage quotients were detected in BRCA1 exons 18 and 23–24 (Fig. 1). Breakpoint characterization of the supposed exon 18 deletion revealed that it was in fact a single base substitution two base pairs away from the ligation site on the long oligonucleotide for exon 18 (c.5215g ? a; R1699Q). A variant at this same position (R1699W) has been detected by MLPA at least once before [28]. This finding once again highlights the importance of confirming the presence of aberrations either using an MLPA confirmation kit or/in addition to long-range PCR, sequencing or RNA/ cDNA analysis. Patient BRC158.1 (Fig. 2) displayed a 46 and 45% reduction in relative peak area (RPA) for exons 23 and 24. Long-range PCR, using primers located in intron 22 and 9.1-kb downstream of the stop codon, produced an expected fragment of 12.3 kb, in addition to a preferentially amplified smaller fragment of approximately 1.2 kb in the patient. The unaffected control had only the single 12.3 kb wild-type fragment (Fig. 3). Sequencing of the

A 1.50

BRC119.1

1.25

RPA

1.00 0.75 0.50 0.25 c c 1a 1b 2 3 5 c 6 7 8 9 10 c 11 11 12 13 14 c. 15 16 17 18 19 c 20 21 22 23 24 c c c

0.00

Probe/Exon

B

BRC158.1 1.50 1.25 1.00 0.75 0.50 0.25 0.00 c c 1a 1b 2 3 5 c 6 7 8 9 10 c 11 11 12 13 14 c. 15 16 17 18 19 c 20 21 22 23 24 c c c

RPA

Fig. 1 Histogram representation of quantitative MLPA analysis of samples showing aberrant profiles. a Analysis of sample BRC119.1 carrying the R1699Q variant in exon 18 which hampers hybridization of the probe. b Analysis of individual presenting with a deletion of exons 23 and 24 (BRC158.1)

deletion fragment revealed that the breakpoints extend from nucleotides g.169527 to g.180579 (Genbank accession no. NG_005905.2), a deletion of 11053 bp. This deletion fuses the AluSc sequence in intron 22 with the AluSq sequence 8-kb downstream of the BRCA1 stop codon, suggestive of an unequal homologous recombination event between these two Alu sequences that share 74% homology. To date, two different deletions of exon 24 [29, 30], and two involving exons 23–24 have been characterized [30, 31]. It is rather interesting to note that the 50 breakpoint of the exons 23–24 deletion (NG_005905.2:g.169527_177 643del) reported by Engert et al. [30] is exactly the same as the one reported in this study (NG_005905.2:g.169527_ 180579del). This nucleotide, situated in the AluSc sequence could possibly represent a hotspot for recombination. Simultaneous deletion of exons 23 and 24 has also been reported in two Italian families [32] and one Spanish family [28], but the breakpoints of these deletions could not be characterized. The two Italian families identified with this rearrangement did not share a common ancestor [32]. cDNA analysis of the exon 24 deletions by Armaou et al. [29] and Engert et al. [30] showed expression from only one allele. The authors found that the removal of the polyA tail region and the 30 -untranslated region of the gene resulted in monoallelic expression, possibly as a result of

Probe/Exon

123


The identification of the DelEx23-24 LGR in this study means that the index case’s daughter (Fig. 2) will now benefit from predictive testing.

BrCa Dx 35

Discussion + BrCa Dx 49

BrCa Dx 30

1kb+ ladder

BRC158.1

WT control

2.5kb ladder

Fig. 2 Pedigree of family positive for the deletion of BRCA1 exons 23-24. Individuals affected with breast cancer are indicated by shaded circles, Dx age at diagnosis

Proportion of BRCA1 LGRs in the BRCA1 mutation spectrum of different populations

12.5kb

2.5kb 2kb Deletion fragment

1kb

Fig. 3 Confirmation of the BRCA1 deletion of exons 23–24 in patient BRC158.1. Ethidium-bromide-stained gel of long-range PCR products of patient BRC158.1 and wild-type (WT) control

non-sense mediated decay [29, 30]. Engert et al. [30] extend this hypothesis to the deletion of exons 23–24, since the same functional regions will be removed. Since the same regions are removed (polyA tail and 30 -UTR) in the deletion reported here, we suggest that the effect of this deletion may be complete loss of the mRNA transcript from the deletion allele. Should the deletion allele however be expressed, crucial functions will be lost, since the BRCT domain will be disrupted and therefore protein–protein interactions which are involved in cell-cycle checkpoints and DNA repair will be ablated [33].

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This represents the first study to screen for LGRs in BRCA1 and BRCA2 in South African breast ovarian cancer patients. MLPA analysis of 66 index cases from 52 HBC/ HBOC families revealed a single BRCA1 rearrangement (Ex23-24del), in a South African family with Greek ancestry. The detection of only one LGR in this South African cohort indicates that such alterations play a small role in the disease in South Africa, while rearrangements in BRCA2 play no role. In the Afrikaner group screened in this study, no LGRs were found (0/36) in either BRCA1 or BRCA2, demonstrating that such mutations play no, or a minimal role in breast/ovarian cancer in this population. This result has direct clinical consequence, as screening for genomic alterations in the BRCA1/2 gene will in all likelihood not be advantageous in the Afrikaner population group.

Comparison of the results generated in this study to those completed in other populations is illustrated in Fig. 4, where the results are given as the proportion of BRCA1 LGRs detected when compared to the overall number of BRCA1 mutations. Conveying the information in this manner is far more informative since the percentage is then independent of selection bias and is thus comparative to other studies [28, 32]. The comparison is only completed for BRCA1, since the general trend in the majority of populations is that BRCA2 LGRs play a minimal role in HBC/HBOC, with the exception of Portugal, where the founder rearrangement c.156_157insAlu (NG_012772.1:g. 8686_8687insAluS) explains more than a quarter of deleterious BRCA mutations [10, 34]. In South Africa, the Afrikaner population appears similar to the Finnish [35–37], French-Canadian [19], and Iranian [20] populations, where BRCA1 LGRs play little/no role in HBC. A stark contrast to this is seen in the Dutch population, where the high proportion of LGRs can be explained by the occurrence of two founder BRCA1 LGRs [18, 38, 39]. Very recently, however, a third BRCA1 Dutch founder (8.2-kb deletion of exons 1–2) has been characterized [40]. The high proportion of LGRs detected in the Hispanic population can also be explained by a single founder deletion of exons 9–12 [41]. The Australian/New Zealand populations also seem to have a large proportion of LGRs, but this is not explained


by a founder effect, but rather by the presence of nonrecurrent LGRs [15]. The proportion of LGRs reported in Italy varies significantly in different reports. According to Montagna et al. [23], BRCA1 rearrangements are responsible for 40% (6/15) of BRCA1 mutations, while Agata et al. [32] state that this value should be 19% (16/86). Buffone et al. [31] found in their study that BRCA1 rearrangements accounted for 10% of the BRCA1 mutations in Italy. Combining this data, 24 BRCA1 LGRs have been detected out of a total of 120 BRCA1 mutations, indicating that LGRs would account for 20% of mutations (Fig. 4). In the Afrikaner group screened in this study, no LGRs were found (0/36) in either BRCA1 or BRCA2, demonstrating that such mutations play no role in breast/ovarian cancer in this population. The Afrikaners are mainly descended from Dutch, German and to a lesser extent, French immigrants to the Cape. The founding Afrikaner population consisted of approximately 90 families by 1687. We therefore expected that the mutations detected within these founding populations would be responsible for at least a portion of the familial breast cancer burden within the Afrikaners. No founder LGRs/small mutations from these populations were detected. Instead, novel small founder mutations unique to the Afrikaner population were detected [24, 42]. In terms of disease-causing BRCA mutations, the South African population seems more similar to the Finnish population, where familial breast cancer mutations are distinct and LGRs are absent [35, 37, 43]. The absence of BRCA2 rearrangements within the South African population is consistent with results from other populations, such as the French-Canadian [19], Finnish [36, 37], Dutch [44], German [45], and Western Danish [46].

Percentage (%)

30

20

10

Afrikaner

Iranian [20]

Fr/Can [19]

Finnish [35-37]

Danish [68]

N. Poland [89]

Portuguese [88]

French [58]

Spanish [28]

Greek [29]

German [30]

Czech [47]

Hispanic [41]

Aus/NZ [15]

Asian [77, 87]

Dutch [18]

Italian [23, 31, 32]

0

Population group

Fig. 4 Percentage of identified BRCA1 mutations for which BRCA1 LGRs are responsible in different populations. Aus/NZ Australia/New Zealand, N. Poland Northern Poland, Fr/Can French Canadian

Recommended nomenclature for naming BRCA1/2 LGRs While designating the genomic breakpoints of the LGR reported here and attempting to determine whether the rearrangement was novel, we came across numerous inconsistencies in the literature. We found that many LGRs are reported as novel, when they have in fact been reported before, while others state their LGRs have been previously reported when they are in fact novel. The cause of these discrepancies is that different groups have used different reference sequences to designate breakpoints, and in some cases the rules of nomenclature (HGVS) appear to be misunderstood. In order to make it easier for authors to determine whether they are describing a novel/previously reported mutation, and to standardize/recommend a universal approach to reporting BRCA1/2 LGRs, we have completed an exhaustive search and compiled lists of all known characterized BRCA1 and BRCA2 LGRs reported to date (Tables 1, 2). Most commonly, authors have chosen L78833.1 and AY436640.1 as the BRCA1 and BRCA2 reference sequences. However, it is now recommended that authors instead use the recently released RefSeqGene records, NG_005905.2 for BRCA1 and NG_012772.1 for BRCA2 (http://www.ncbi. nlm.nih.gov/RefSeq/RSG/). Please note that the BRCA1 reference sequence has very recently been updated to the second version NG_005905.2. The first version of the reference sequence only extended 11.8 kb 50 of the BRCA1 start codon, and 6.3-kb downstream of the stop codon. This meant that a number of LGRs could not be classified using only one reference sequence. It is for this reason that we requested that the reference sequence be extended to include 93-kb upstream of BRCA1 (including NBR2, wBRCA1, and NBR1) and 20-kb downstream of BRCA1. It is therefore now possible to describe all BRCA1 LGRs using only one reference sequence. The availability of the upstream region should also make it easier for authors to characterise deletions involving the 50 region of the gene, since designing primers for this region and characterizing such LGRs appears to have been a problem in the past. We have converted the breakpoints of all characterized BRCA1/2 LGRs to the RefSeqGene records, NG_005905.2 or NG_012772.1, in accordance with the recommended HGVS nomenclature [27]. With this we hope to assist in standardizing the reporting of BRCA1/2 LGRs. Please note that it is required to give the version number in addition to the accession number when stating the reference sequence (Use NG_005905.2, and not only NG_005905). Apart from the use of different reference sequences for naming LGRs, discrepancies occur in terms of misunderstanding nomenclature.

123

123

Deletion BPs occur in 23 bp NG_005905.2:g.61201_98134del 36,934 bp deletion regions of perfect homology at [Regions of homology extend g.34339–34361 in BRCA1 and from g.61178 to 61200 in 71274–71296 in wBRCA1 wBRCA1 and g.98112 to 98134 (AC060780) in BRCA1]

Deletion BPs occur in a region of NG_005905.2:g.61422_98355del 36,934 bp deletion 188 bp of perfect homology [Regions of homology extend between g.34118–34305 in from g.61234 to 61421 in BRCA1 and g. 71053–71240 in wBRCA1 and g.98168 to 98355 wBRCA1 (AC060780) in BRCA1]

Deletion BPs occur in a region of NG_005905.2:g.85341_99155del 13,815 bp deletion 23 bp of perfect sequence [Regions of homology extend identity between g.33319– from g.85317 to 85340 in 33342 in BRCA1 and g.47133– wBRCA1 and g.99132 to 99155 47155 in wBRCA1 (AC060780) in BRCA1]

1–2

1–2

Deletion with BPs at NG_005905.2: 17:38543865 and 17:38519865 g.79662_103662del (UCSC sequence)


AC060780.18: g.123637_L78833: g.80466del

Deletion with BPs at 17:38538510 and 17: 38451657 (UCSC sequence)

1-3

1–12

1–22

1–23

NG005905.2: g.85019_171870del

NG_005905.2: g.8836_169713del

NG_005905.2:g.62439_99207del

AC060780: g.33267_70035del

1–2

NG_005905.2: g.86268_94435delins AAAAAAAAA

AC060780: g.38037_46204delins9

1–2

Deletion removes NBR2, the BRCA1 promotor and results in a chimeric gene*


wgene/gene HR

wgene/gene HR

wgene/gene HR

86,852 bp deletion

160,878 bp deletion

88,551 bp deletion

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Deletion, with size reported as 23 Alu/Alu HR 395 bp, but according to the NG005905.2 seq, size of deletion would be 24001 bp

36 769 bp deletion

Dutch

2

4

No transcript

Deletion removes NBR2 and 18 of the 19 NBR1 exons, wBRCA1 and over 95% of the BRCA1 sequence

No transcript

No transcript

1

1

1

1

Hybrid RNA is expressed 1 containing exons 3–24 from WT BRCA1 and exons 1–2 from wBRCA1

Authors suggest that no transcript will be present

Irish

French

Irish

Norwegian

German

Dutch

MLPA, cDNA analysis ? LRPCR

QMPSF, DNA combing ? LRPCR



MLPA, cDNA analysis, LR-PCR

MLPA, Haplotype, LOH, a-CGH analysis, LR-PCR


MLPA, LR-PCR

MLPA, LR-PCR

Spanish

Czech

1

Haplotype, Southern analysis ? PCR

German

1

Southern analysis ? PCR

Southern analysis ? LRPCR

MLPA, LR-PCR

MLPA, aCGH, LRPCR

MLPA, LR-PCR, cDNA analysis


American

British

1

American

German

1

1

Italian

American

1

1

# Famsf Nationality/ Detection ancestry methodh of familiesg

Transcription is eliminated 1 since both transcription start sites as well as exon 2 are 1 removed.


wgene/gene HR

Alu/Alu HR

Predicted effecte

Type of sequence at breakpoint and mechanism of rearrangementd

8,168 bp deletion, and an wgene/gene HR insertion of 9 A’s in the deleted region

36,934 bp deletion

1–2

[Regions of homology extend from g.60864 to 61100 in wBRCA1 and g.97798 to 98034 in BRCA1]

NG_005905.2:g.61101_98034del

Deletion BPs occur in 2 regions of 237 bp that share 100% homology from g.34439 to 34675 in BRCA1 and g.71374 to 71610 in wBRCA1 (AC060780)

1–2

Size of LGRc

HGVS nomenclatureb

Exons Mutation with breakpoints as reported (ref seq #)a

Table 1 List of characterized BRCA1 rearrangements

[13]

[64]

[13]

[13]

[30]

[40]

[63]

[17]

[47]

[45]

[52]

[62]

[40]

[30]

[23]

[62]

Reference


L78833:g.12977 ins10 del1039

Deletion BPs at 17:38520262 and NG_005905.2:g. Reported as a deletion Non-alu/non-alu NHE 17:38521304 (UCSC sequence) 102223_103265delins7 of 1049 bp and an as well as a 7 bp insertion. insertion of 7 bp. [Walsh et al. state this deletion has According to the been previously reported (without a NG_005905.2 reference), but according to this sequence the size of exhaustive search the only the deletion would previously reported complex LGR be 1043 bp involving this exon is the one reported by Payne et al. [66]. The breakpoints of the deletion when translated from the UCSC sequence to the NG_005905.2 sequence are g. 102223 and g.103265. Correct nomenclature cannot be given, since the sequence of the insertion is not indicated in the paper]

L78833:g.8097_22733del

L78833:g.11967_29213dup

IVS2 ? 7220_IVS16 ? 717del46583 [46] NC_000017:g. 8655_55240del46586 [56]

3

3

3–5

3–8

3–16 [Although these deletions are reported to differ in size by 3 bp, they do in fact have the same breakpoints according to the electropherograms supplied by the authors. According to the NG_005905.2 sequence the breakpoint region has a 26 bp region of complete homology between g.101187 and 101212 in intron 2 and g.147773 and 147798 in intron 16]

NG_005905.2:g.101213_147798del

NG_005905.2:g.101216_118464dup

NG_005905.2:g.97346_111983del

[Derived from electropherogram provided by Payne et al. [66]]

NG_005905.2: g.102227_103265delinv: g.102215_102224

[Although deletion is reported using the refseq NG_005905.1, from the electropherogram provided, the deletion actually extends from g.11748 to g.20860 in this sequence]

NG_005905.2:g.93770_102882del

NG_005905.1: g.1238_10350del Alu/Alu HR

46 586 bp deletion

17 249 bp duplication

14 638 bp deletion

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

1

Not stated. possibly results in a stop at codon 27

Stop at codon 186

Not stated. could possibly result in an In-frame deletion of 44 aa

Stop at codon 27

9

2

1

1

?

1

Danish

Danish

French

Spanish

English

American

Italian

British

MLPA ? LR-PCR

MLPA ? LR-PCR

Combing ?LR-PCR

MLPA ? LR-PCR

MLPA, cDNA analysis

cDNA analysis ? LRPCR




Either absence of expression due to 1 deletion of the start codon, or if the in-frame ATG at the start of exon 5 functions as a translation initiation codon, the first 47 aa of BRCA1 will be deleted, removing the Zinc-finger RING domain.

Start codon is deleted

Predicted effecte

Stop at codon 27 Deletion of 1,039 bp Non-alu/non-Alu NHE. and an inverted 50 BP lies within exon 3 duplication of 10 bp located in exon 3

9,113 bp deletion

Alu/non-Alu NHE

2–3

2,535 bp deletion

NG_005905.2: g.93041_95575del

L78833:g.3792_6326del


2

Size of LGRc

HGVS nomenclatureb


Table 1 continued

[56]

[46]

[67]

[63]

[13]

[66]

[65]

[49]

Reference


123

123

Deletion BPs lie in a 15 bp region NG_005905.2: of complete sequence identity g.110447_115470del between g.22035–22049 in [Region of 15 bp homology intron 4 and g.17011–17025 in extends from g.110432 intron 7 (AC060780) to 110446 and g.115456 to 115470]

NC_000017:g.18296_23289del [c.136-623_441 ? 1959del]

L78833:g.21716_53298del

Deletion starts at position IVS7-1129 and removes 1458 bp

L78833: g.26181_29711del

L78833: g.25529_31240del

Deletion BPs at 17:38506245 and 17:38502281 (UCSC sequence)

5–7

5–7

5-14

8

8

8

8-9

NG_005905.2: g.117280_121246del

NG_005905.2: g.114779_120492del

NG_005905.2: g.115430_118963del

[Please note: size of del will be different here depending on which sequence is used. Here we first used the L78833.1 sequence and translated that into the NG_005905.2 sequence, since the L78833.1 sequence was most likely used by the authors to determine the BPs]

NG_005905.2: g.116973_118432del

NG_005905.2: g.110966_142550del

NG_005905.: g.103629_108623del

NG_005905.2: Deletion BPs lie in a region of g.111421_111664del 9 bp that have complete homology between g.21060– [Region of 9 bp homology 21068 in intron 4 and g.20816– extends from g.111412 to 20824 in intron 5 (AC060780) 111420 and g.111656 to 111664]

HGVS nomenclatureb

5


Table 1 continued

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Stop at codon 164

Stop at codon 152

?

1

Not stated. 1 Will possibly introduce a stop at codon 152

Not stated, but 1 will possibly introduce a stop at codon 152

African American

German

German

Dutch

Czech German

4 1

In-frame Non-alu/non-Alu deletion (NHE). The L1 of 1450 aa sequence at the 50 BP does not correlate with any repeat motif at the 30 BP

Danish, with British Ancestors

German Spanish

1

German

1

1

German

1

# Famsf Nationality/ ancestry of familiesg

1

Stop at codon 163

In-frame deletion of 26aa

Predicted effecte

Predicted to result in a stop at codon 163

Alu/Alu HR

Alu/Alu HR

Non-Alu/non-Alu HR. BPs within region of 100% homology, but not located in Alus


Deletion size reported as 3936 bp, but Non-Alu/Non-Alu according to NG_005905.2 seq, del would NHE be 3967 bp

5 714 bp deletion

3,534 bp deletion

1,460 bp deletion

31,585 bp deletion

4,995 bp deletion

5,024 bp deletion

244 bp deletion

Size of LGRc


MLPA, cDNA ? nested PCR


MLPA ? LR-PCR


MLPA ? LR-PCR

MLPA, LR-PCR


MLPA ? LR-PCR

MLPA, LR-PCR


Detection methodh

[13]

[30]

[30]

[18]

[30]

[47]

[56]

[63]

[45]

[30]

[45]

Reference


23,767 bp deletion 65,520 bp deletion

NG_005905.2: g.116318_140084del

L78833: g.27069_50831del

Deletion BPs at 17:38507180 and NG_005905.2: 17:38441660 (UCSC sequence) g.116347_181867del

Deletion BPs are located between NG_005905.1: nucleotides 29,624 and 29,702 g.118955_133611del in intron 8 and nucleotides [Regions of homology extend 44,280 and 44,358 in intron 12 from g.118876 to 118954 and (L78833) g.133533 to 133611]

L78833:g.29197_65577del

L78833:g.34845_41405del

c.1739_1740insAlu

8–24

9–12

9–19

Part of 11 - 12

11

6 562 bp deletion

36 381 bp deletion

Spanish Spanish

1 1

Czech

1 Non-Alu/non-Alu NHE. This deletion removes almost 50% of the coding sequence (72% of exon 11 is The L1 repetitive removed). Possible stop at codon 557. sequence present near Authors suspect that aberrant splicing of the 30 breakpoint does mRNA may extend a further defect on the not correlate with any protein repeat motifs at the 50 breakpoint.

Belgian

Italian

An Alu element from the AluSp/AluSq family 2 is inserted in exon 11. Effect of the insertion was investigated on mRNA level, but results were inconclusive.


MLPA ? LR-PCR


MLPA ? LR-PCR

Combing ? LRPCR

Combing ? LRPCR


Southern analysis

PTT, Southern analysis, cDNA analysis ? LRPCR

PTT ? LR-PCR

MLPA ? LR-PCR



4 Hispanic MLPA, cDNA and 1 analysis ? LRAfrican/ PCR Native American

German

1

5

?

British

French

1

1

French

1

Italian

Stop at codon 184

Stop at codon 190

Stop at codon 148

Stop at codon 150

French

American

1

1 Stop at codon 150

American


Rearrangement results in 2 alternatively 2 spliced mutant transcripts. One lacks exon 8 –9 (stop at codon 164) and the other lacks exons 8 –10 (stop at codon 157)

Predicted effecte

1

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR


NG_005905.2: Insertion of Retrotransposong.124192_124193insAlu an Alu mediated insertion. [Correct HGVS nomenclature element cannot be given, since the seq of of the inserted segment has not unknown been determined. See size discussion on nomenclature in text.]

NG_005905.2: g.124097_130658del

NG_005905.2: g.118448_154828del

14 657 bp deletion

23,767 bp deletion

8–13

NG_005905.2: g.116216_139982del

L78833: g.26967_50729del

8–13

7,090 bp deletion

NG_005905.2: g.114552_121641del

L78833: g.25302_32389del

8–9

Size of LGRc

HGVS nomenclatureb

Mutation with breakpoints as reported (ref seq #)a

Exons

Table 1 continued

[50]

[47]

[32]

[23]

[41]

[13]

[49]

[63]

[28]

[58]

[59]

[70]

[69]

[57]

Reference


123


L78833:g. 33450_56562del

L78833: g.44513_48347del

L78833:g.44369_50449dup x (17:38483825 and 17:38489905)

L78833:41220_49682dup

Exons

11–15

13

13

13

Table 1 continued

123 NG_005905.2: g.130473_138935dup

NG_005905.1: g.133622_139702dup

NG_005905.2: g.133766_137600del

NG_005905.2: g.122702_145814del

HGVS nomenclatureb


6,081 bp duplication

3,835 bp deletion

23,113 bp deletion

Size of LGRc

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Non-Alu/Alu NHE


Not stated. Will possibly introduce a Stop at 1398

Stop at codon 1460

Stop at codon 1398

Australian Canadian British American Belgium British British German German Dutch W/N Eu C/E Eur/Lat/Car Af Am German Eng/Ir, NS

1 1 6 2 1 5 1 1 1 1 36

British Danish German

6 1 4

Chinese

Northern European

?

1

German

2

12

1

1

2

1

Portuguese

1

German

1 American

NS

1

3

Dutch

6

Spanish


Not stated. Could 1 possibly cause an in-frame deletion of exons 11–15

Predicted effecte

MLPA, LR-PCR

MLPA ? PCR

MLPA, LR-PCR

MLPA, Mutation specific PCR


MLPA ? LR-PCR

Mutation-specific multiplex PCR

MLPA ? PCR

QMPSF ? PCR

PCR

PCR

Semiquantitative PCR

PCR

cDNA ? LR-PCR

MLPA, LR-PCR


Southern analysis ? LR-PCR

MLPA ? LR-PCR

Detection methodh

[75]

[30]

[56]

[49]

[13]

[45]

[71]

[18]

[74]

[73]

[69]

[72]

[21]

[53]

[30]

[71]

[38]

[28]

Reference


Deletion BPs lie within 22 bp region of complete homology between g.44330–44351 and g.54740–54761 (L78833)

c.4186-1593_4676- 1465del. This NG_005905.2: 11 466 bp translates to g.44563_56029del g.133816_145281del deletion on the L78833.1 sequence Please note: size of del will be different here depending on which sequence is used. Here we first used the L78833.1 sequence and translated that into the NG_005905.2 sequence, since the L78833.1 sequence was most likely used by the authors to determine the BPs

L78833:g.47840_52790del

L78833:g.51482_71368del

Deletion BPs lie in a 54 bp region NG_005905.2: 26 448 bp of identity between g.50524– g.139830_166277del deletion 50576 and g.76967–77020 [Deletion BPs lie in a region of (L78833) identity between g.139777– 139829 and g.139829–166277]

13–15

13–15

14

14–19

14-20

NG_005905.2:g. 140734_160619del

NG_005905.2: g.137093_142042del

[Deletion BPs are located between g.133583–133604 in intron 12 and g.143992–144013 in intron 15]

NG_005905.2: g.133605_144013del

NG_005905.2: g.133630_145231del

Exactly the same size as the deletion reported by Gad et al. [76], but the breakpoints are 22 bp proximal to the deletion reported in the American family

13–15

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR


19,886 bp deletion

Alu/Alu HR

Non-Alu/Alu NHE

4 950 bp deletion Alu/Alu HR

10,409 bp deletion

11,602 bp deletion

11,602 bp deletion

Deletion BPs are located between NG_005905.2: g.44377–44397 in intron 12 and g.133651_145252del g.55980–56000 in intron 15 [BPs located between g.133630– (L78833) 133650 in intron 12 and 145232–145252 in intron 15]

Size of LGRc

13–15

HGVS nomenclatureb


Exons

Table 1 continued

1

2

1

1

1


Irish

Five-site rearrangement panel (Myriad Genetics)


6 W/N Eur; 1 NS

British

Haplotype analysis, LR-PCR

Haplotype, cDNA analysis ? LRPCR

MLPA ? LR-PCR

MLPA, LR-PCR

MLPA ? LR-PCR


MLPA ? LR-PCR

Combing, LR-PCR and cDNA analysis

Detection methodh

18 W. Eur, 1 Ir, 2 NS

Italian

Spanish

Korean

Indian

German

Danish

1 1

American

1


Predicted to truncate the protein 21 at codon 1523 (Ward et al., 2005). 7 However, Walsh et al. [13] report that this deletion will result ? in removal of 307amino acids

Not stated but could possibly introduce a stop at codon 1454


Possible stop at codon 1437

Predicted to result in a stop at codon 1437

Stop at codon 1437

Stop at codon 1437

Predicted effecte

[81]

[13]

[71]

[80]

[79]

[28]

[78]

[77]

[30]

[46]

[76]

Reference


123

123

Deletion BPs lie in a 46 bp region NG_005905.1: 5,629 bp deletion of identity between g.53030– g.142328_147956del 53075 and g.58659–58704 [BPs lie between g.142282– (L78833) 142327 and g.147911–147956]

L78833:g.53092_76493delins23

L78833:g.56074_58704del

L78833:g.56990_75331del

c.1126 ? 1370_1919-385del

L78833.1:g.67689_68701del NG_005905.2: (According to Walsh et al. [13] g.149711_150723del BPs occur at 17:38472804 and 17:38473817; UCSC sequence)

L78833:g.58759_61875del NG_005905.2: According to Walsh et al. [13] g.48011_151126del BPs occur at 17:38472400 and 17:38475516 (UCSC sequence)

L78833:g.58530_61209del NG_005905.2: According to Walsh et al. [13] g.147782_150460del BPs occur at 17:38473067 and 17:38475745 (UCSC sequence)

NC_000017:g.55023_61749del

15–20

16

16–20

16–23

17

17

17

17–18

NG_005905.2: g.147555_154281del

[Deletion BPs occur in 2 regions of 21 bp that share 100% homology from g. 45003 to 145023 and g.171797–171817]

NG_005905.2: g.145024_171817del

NG_005905.1: g.146242_164582del

NG_005905.2: g.145326_147956del

[Correct nomenclature cannot be given, since seq of insertion is not indicated]

NG_005905.2: g.142344_165740delins23

Alu/Alu HR

Alu/Alu HR


6 727 bp deletion

2 679 bp deletion

3,116 bp deletion

1 013 bp deletion

26 794 bp

18,341 bp deletion

2,631 bp deletion

Non-Alu/non-Alu NHE

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

23,397 bp deletion Alu/non-Alu NHE and 23 bp insertion

2,998 bp deletion

15–16

NG_005905.2: g.142354_145351del

L78833:g.53102_56099del

Size of LGRc

15

HGVS nomenclatureb


Exons

Table 1 continued

Predicted to introduce a stop at codon 1719

Stop at codon 1672

Stop at codon 1672

Stop at codon 1672

In-frame deletion of exons 16–23

Stop at codon 1628

Stop at codon 1574

Stop at codon 1500


Predicted to stop at codon 1509

Predicted effecte

1

Danish with Iranian ancestry

German

?

German

6

German

Irish, Swedish, Norwegian

?

1

German Italian

1 2

Italian

2

American German

1

Spanish

Italian

German

British

French

French

?

1

1

2

1

1

1


MLPA, LR-PCR


MLPA ? LR-PCR

MLPA ? LR-PCR

MLPA, CDNA analysis ? LRPCR


MLPA ? LR-PCR


MLPA, CDNA analysis ? LRPCR

cDNA ? LR-PCR

MLPA ? LR-PCR,




QMPSF ? LR-PCR


Detection methodh

[56]

[13]

[30]

[30]

[13]

[32]

[45]

[60]

[13]

[16]

[83]

[32]

[30]

[49]

[82]

[70]

Reference


L78833: g.58668_66883del; g.68668_68669in s61320_61740; g.68669_72379del

L78833:g.62115_66940del

L78833:g.63651_65590del

L78833:g.63335_70579 delinsAC060780: g.99286_99312

Duplication with BPs at NG_005905.2: 17:38472226 and 17:38466304 g.151301_157223dup (UCSC sequence)

L78833:g.63859_72516dup

According to Walsh et al. [13] BPs NG_005905.2: occur at 17:38458944 and g.160599_164583del 17:38462928 (UCSC sequence). BPs not stated in Carson et al. [85], but Mazoyer [52] references Carson’s deletion as having the following breakpoints: (L78833:g.71348_75332del) Palanca et al. state their exon 20 deletion is the same as the one reported by Carson et al. [85]

18–19

18–19

18–19

18–19

18–20

20

NG_005905.2: g.153110_161767dup

NG_005905.2: g.152856_159830delins: g.33163_33189

NG_005905.2: g.152902_154841del

NG_005905.2: g.151366_156191del

NG_005905.2: g.147920_156134del; g.157919_157920ins g.150571_150991; g.157920_161630del

[IVS16 ? 927–IVS19 ? 1681 translated to the L78833 seq:g.58730_67081]

17–20

Alu/Alu HR

Type of sequence at breakpoint and mechanism of rearrangementd Not stated, but will most probably result in an Inframe triplication

Predicted effecte

Alu/Alu HR

Alu/Alu HR

3,985 bp deletion



Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

7,245 bp deletion, Alu/Alu HR 27 bp insertion from NBR1 intron 1

1,940 bp deletion

4 826 bp deletion


Stop at codon 1769

In-frame duplication of 40aa

Premature stop created. Not stated where, but could possibly be at codon 1693

Authors predict a stop at codon 1693

Stop at codon 1693

8,215 bp deletion, a Alu/Alu HR (6 different In-frame deletion of exons 421 bp insertion and Alu elements are 17–20 a 3 711 bp deletion involved)

8 351 bp triplication

NG_005905.2: g.147982_156332[3]

Triplication starts at IVS19 ? 1681 (antisense strand) and includes 8352 nt

17–19

Size of LGRc

HGVS nomenclatureb


Exons

Table 1 continued

Italian

1

Canadian

French

Spanish

?

1

French

African American

German

1

1

?

1

Czech

Italian

1

MLPA ? Southern analysis ?LRPCR

Detection methodh


MLPA, cDNA analysis

PTT ? Southern analysis ? LRPCR

Combing, cDNA analysis ? LRPCR


MLPA ? LR-PCR

MLPA ? LR-PCR

MLPA, real-time qPCR ? LR-PCR


PTT, Southern American with analysis, cDNA Anglo-Saxon and analysis ? LRAsh Jewish descent PCR

Dutch

1

1

1


[63]

[13]

[85]

[58]

[13]

[30]

[47]

[31]

[23]

[84]

[18]

Reference


123

123

L78833:g.71618_74863del

L78833:g.67058_75763dup

L78833:g.68764_75792del

Deletion BPs occur in 23 bp NG_005905.2: regions of perfect homology g.160420_164592del from g.71146 to 71168 in intron [BPs located between g.160397– 19 and g.75319 to 75341 in 160419 and g.164570–164592] intron 20

L78833:g.65740_73887del

Deletion starts at IVS19-2840 and NG_005905.2: includes 11395 nt g.158008_169402del

Deletion BPs at 17:38465050 and NG_005905.2: 17:38453698 (UCSC sequence) g.158477_169829del

L78833:g.76887_80317del

L78833:g.77128_80906del; g.72835_73070ins

Tandem duplication of 7654 nt starting at IVS23 ? 343

20

20

20

20

20

20–22

20–22

21–22

21–22

21–23 Please note: size of del will be different here depending on which sequence is used. Here we first used the L78833.1 sequence and translated that into the NG_005905.2 sequence, since the L78833.1 sequence was most likely used by the authors to determine the BPs.

NG_005905.2: g.163035_170685dup

NG_005905.2: g.166375_170153delins: g.162086_162321

NG_005905.2: g.166134_169564del

NG_005905.2: g.154991_163138del

NG_005905.2: 158015_165043del

NG_005905.2: g.156309_165014dup

NG_005905.2: g.160869_164114del

NG_005905.2: g.157279_161606del

L78833:g.68028_72355del

20

HGVS nomenclatureb


Exons

Table 1 continued


3,779 bp deletion ?236 bp insertion

3,431 bp deletion

11,353 bp deletion

11,395 bp deletion

8,148 bp deletion

4,173 bp deletion

7,029 bp deletion


3 246 bp deletion

4 328 bp deletion

Size of LGRc

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Non-Alu/Alu NHE

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Non-Alu/Alu NHE. 5’ break is in exon 20

Alu/Alu HR


1

1

1

Not stated, but will possibly introduce a stop at codon 1770

In-frame deletion of 43 aa removes substantial part of the second BRCT domain

In-frame deletion of exons 21 and 22 (43 aa)


1

Dutch

Czech Czech

2

American of Irish descent

English

1

1

?

Dutch

German

Greek

Czech

Italian

Greek

1

1

Greek

Italian

1

1


Not stated, but will probably 1 result in an In-frame deletion of 71 aa


Stated as unknown, but will possibly also result in an In-frame deletion of 28aa


In-frame duplication of 28 aa

Resultant protein has 20 aa of the BRCT-linker domain missing. This deletion should also create a stop at codon 1743.


Predicted effecte

MLPA, Southern analysis ? LRPCR

PTT ? LR-PCR

MLPA ? LR-PCR

PTT, Southern analysis, cDNA analysis ? LRPCR


MLPA ? LR-PCR


QMPSF ? LR-PCR

MLPA ? LR-PCR


PCR



Detection methodh

[18]

[48]

[47]

[84]

[13]

[18]

[30]

[29]

[47]

[32]

[29]

[86]

[23]

Reference


L78833:g.79505_80014del

Reported as c.5333-198_5387del, NG_005905.2: but according to the g.168592_168844del NM_007294.2 sequence which the authors used, it should actually be c.5533198_5587del

L78833:g.80363_83763del

L78833:g.80280_88398del

L78833:g.80280_91331del

L78833:g.82651_87079del

L78833:g.82974_84478del

22

22

23– 24

23– 24

23– 24

24

24

NG_005905.2: g.172220_173725del

NG_005905.2: g.171898_176324 delinsCACAG

NG_005905.2: g.169527_180579del

NG_005905.2: g.169527_177643del

NG_005905.2: g.169610_173009del

NG_005905.2: g.168752_169261del


HGVS nomenclatureb

21– 24


Table 1 continued

1,506 bp deletion

Stop at codon 1804

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Dutch German

? 3

2

1

Non-Alu/Non-Alu NHE. Monoallelic expression 50 BP lies within exon 24

1

1

1

Monoallelic expression

Predict Monoallelic expression based on results provided by Engert et al. [30]

Authors predict Monoallelic expression but in Table 2 of the paper they state it will result in a stop

Authors predict the introduction of 55 aa encoded by unspliced IVS22 before a new stop is encoded

W/N Eur

5

German

Greek

Greek

German

Italian

Spanish/ Central African

German

1

1

Dutch

14

Irish


QMPSF, cDNA analysis ? LRPCR

MLPA ? LR-PCR

MLPA ? LR-PCR

MLPA, real-time qPCR, LR-PCR





PCR

Southern analysis, cDNA analysis ? LRPCR



Authors indicate that the deletion results in a stop at ? codon 1778. From the cDNA sequence it does however appear as if it should stop at 1759, the last codon of exon 20. Should splicing be hindered by this deletion, the next stop would then be at codon 1767

Predicted effecte

Non-Alu/Non-Alu NHE Stop at codon 1832

Non-Alu/Alu NHE

Alu/Alu HR


4427 bp Alu/Non-Alu NHE deletion together with 5 bp insertion

11,053 bp deletion

8 117 bp deletion

3 400 bp deletion

253 bp deletion

510 bp deletion

19,245 bp deletion

Size of LGRc

[30]

[29]

Current study

[30]

[31]

[83]

[30]

[13]

[71]

[73]

[38]

[13]

Reference


123

* This deletion results in a chimeric gene that comprises exons 1A, 1B, and 2 of wBRCA1 fused to exons 3–24 of BRCA1

aa amino acids

? No. of families identified with this LGR was not indicated

Nationality/ancestry of mutation positive families. W.E Western European, NS not specified, W/N Eur Western/Northern European, C/E Eur Central/Eastern European, Lat/Car Latin American/Caribbean, Af Am African American, Ir Irish

Number of families identified with a particular rearrangement

Methods used to detect rearrangement: LR-PCR long-range PCR, MLPA multiplex ligation-dependent probe amplification, PTT protein truncation test, QMPSF quantitative multiplex PCR of short fluorescent fragments, aCGH array comparative genomic hybridization, LOH loss of heterozygosity analysis

h

g

f

Type of sequence at breakpoint and mechanism of rearrangement: HR homologous recombination; NHE non-homologous event

Effect particular rearrangements is predicted to have on the transcript or protein. If cDNA was not completed by the authors, the effect of the LGR is predicted using the NM_007294.2 sequence e

d

Size of LGR—the size indicated in this column may be different to that reported, since there may be differences in the reference sequences used. Please note that in this column, the RefSeqGene record, NG_005905.2 was used to calculate these values

HGVS nomenclature—reported breakpoints, which are shown undera, have been converted, using the RefSeqGene record, NG_005905.2, and the recommended HGVS nomenclature

123

c

b

a Clone sequences utilized for identification of BRCA1 breakpoints: AC109326.11, AC060780, NC_000017, L78833.1, and the UCSC sequence [obtained from the Genomic locale from the Human Genome Browser, May 2004 assembly (http://genome.ucsc.edu/)]

Table 1 continued


In the following section, we attempt to explain in more depth and clarify any misunderstandings regarding the HGVS nomenclature as described by den Dunnen and Antonarakis [27]. First, when describing a deletion, the incorrect bases are often used as the breakpoints. The 50 breakpoint is the first base pair which is actually deleted, and the 30 breakpoint is the last base pair which is deleted. This becomes complicated in instances where the breakpoints occur in two regions that share 100% homology. In such cases it is impossible to know exactly where the breakpoint occurred, and while it is true that the most 30 -base should be used as the breakpoint, it would be incorrect to simply use the most 30 -base of each of the regions displaying homology (refer to Fig. 5 for a detailed explanation). The breakpoints in this hypothetical gene lie in a 19-bp region of complete homology between nucleotides g.15831–g.15849 and g.16051–g.16069. Using the most 30 -bases as the breakpoints, one would therefore assume that the deletion should be named g.15849– g.16069del. This is however incorrect if one examines the electropherogram provided (Fig. 5b). The first deleted base is actually the base following the most 30 -base at the 50 -homologous region, i.e., g.15850. The deletion should therefore be called L13789.4:g.15850_g.16069del. Also, when naming deletions, the number of base pairs deleted should not be indicated, only ‘‘del’’ should be denoted after giving the breakpoints, thereby preventing confusion often caused by including unnecessary information (i.e., often the size of the deletion is miscalculated, causing confusion when the same deletions are reported). In instances where both a deletion and an insertion occur at the same site, the deletion should be referred to first, followed by the insertion. For example, if we refer to the same hypothetical deletion above and if this deletion occurred together with an insertion of 4 bp, the mutation would be called L13789.4:g.15850_g.16069delinsAGCT, because the insertion occurred at the same position in which the deletion occurred. It is important to indicate which basepairs are inserted. If a large number of basepairs are inserted, it becomes difficult to indicate the exact sequence. In such instances, the nucleotide numbers and accession number of the inserted sequence should be indicated. There is not an exact limit/cut-off in the case of insertions, but den Dunnen (personal communication) suggests that one should choose that option that gives the shortest unequivocal description. This may mean that the origin of the inserted sequence must be determined, in order to refer to a GenBank record containing it, or alternatively one will have to submit the identified sequence to GenBank and refer to that record. In instances, where large insertions occur on their own, the two nucleotides flanking the insertion should be indicated, not just the nucleotide preceding the insertion. If the inserted

AY436640:g.8730_24909dup

AY436640:g.12323_26644del

7069 ? 686bpdel6.2kbins(A)‘60 (?)

Deletion BPs occur in 11 bp NG_012772.1:g.31338_39284del 7,947 bp regions of perfect homology deletion [BPs located between g.31327–31337 and from g.27555 to 27565 in intron g.39274–39284] 11 and g.35511 to 35521 in intron 13 (AY436640.1)

AY436640.1:g.27422_36313del

AY436640.1:g.37838_54065del

AY436640.1: [g.42188insTAAATACTGAC; g.42189_47824del]

8-part of exon 11

12–13

12–13

12–13

14–18

15–16

NG_012772.1: g.45948_51583delins TAAATACTGAC

NG_012772.1:g.37848_57818del

5 635 bp deletion, 11 bp insertion

16 209 bp deletion

8,883 bp deletion

6,212 bp deletion and an insertion of * 60 Adenine residues at breakpoint junction

NG_012772.1: g.31404_37615delins(A)*60

NG_012772.1:g.31194_40076del

14 318 bp deletion


NG_012772.1:g.16099_30416del

NG_012772.1:g.12507_28681dup

1

?

In-frame duplication of exons 4 to 1 the first 56% of exon 11

Alu/non-Alu NHE

Alu/Alu HR

Alu/Alu HR

Alu/Alu HR

Not stated, but may result in premature stop at codon 2496




Alu/non-Alu NHE. The 5’ Stop at codon 2311 deletion BP (intron 11) is in the 3’ polyA tail of an Alu element, where a track of approximately 60 adenine nucleotides are inserted

1

1

1

1

1

PCR

Portuguese

French

French

French

French

Israeli

Italian

QMPSF, LR-PCR

QMPSF, LR-PCR

QMPSF, MLPA, LR-PCR

QMPSF, LR-PCR

Southern analysis and LR-PCR


MLPA, LR-PCR

3-step PCR (PCR, nested PCR, RT PCR)

Portuguese

Chinese

Southern analysis, LR-PCR

cDNA analysis, LRPCR


Portuguese

Swedish

English

#Famsf Nationality/ Detection ancestry of methodg families

1 Alu element of the AluYa5 subtype is inserted into exon 3, resulting in the In-frame 17 deletion of exon 3 in the transcript. A transcriptional activation domain is therefore 14 removed

In-frame deletion

No transcript

Predicted effecte

Non-Alu/non-Alu. No repeat Part of exon 7 is removed from 1 sequences were detected at the transcript. The first 79 nts of the ends of the exon 7 are joined to the last 301 recombining sequences. 50 nts of exon 11. End result is BP in exon 7, 3’ BP in termination at codon 206. (94% exon 11 of exon 11 is deleted)

Non-Alu/Non-Alu NHE. 5 BP has homology to a L1 repeat

0

Retrotransposon-mediated insertion

4-part of exon 11

NG_012772.1:g.8686_8687insAluS [Correct HGVS nomenclature cannot be given, since the seq of the inserted Alu segment must be indicated. See discussion on nomenclature in text]

*0.3 kb insertion

c.156_157insAlu

Non-Alu/Non-Alu NHE. 50 BP is in exon 3.

Alu/Alu HR

Type of sequence at breakpoint & mechanism of rearrangementd

5,064 bp deletion and 4 bp insertion

3

[Correct HGVS nomenclature cannot be given, since the seq of the inserted segment is not indicated in the article.]

Nt504del5068insCCAT (U43746) NG_012772.1:g.8807_13871delinsCCAT

2,353 bp deletion and 12 bp insertion

Size of LGRc

3

NG_012772.1:g.4093_6446delins12

HGVS nomenclatureb

Deletion BPs at 13:31786709 and 13:31789062 (UCSC sequence)

x


1-2

Exons

Table 2 List of characterized BRCA2 rearrangements

[9]

[9]

[9]

[12]

[14]

[7]

[77]

[34]

[10]

[50]

[11]

[13]

Reference


123

123

Reported as c.7436-270_7805 ? 691del, but according to the NM_007294.2 sequence which the authors used, it should actually be c.7662-270_ 8032 ? 691del

AY436640 g.45138_g55975del

15–16

17–18

AY436640:g.56447_61399del

Deletion with BPs at NG_012772.1:g.65941_67441del 13:31848537 and 13:31850057 (UCSC sequence)

AY436640:g.65122ins346

20

21

22 346 bp insertion

Stop at codon 2957

Alu/Alu HR

1

?

1

1

?

Japanese

Hungarian

Italian

Danish

Dutch, German

Italian

Spanish

SSCP



MLPA, LR-PCR




#Famsf Nationality/ Detection ancestry of methodg families

Retrotransposon-mediated Results in alternative splicing 1 Insertion. The presence of a and skipping of exon 22 in the 64 bp polyA track and an transcript Stop at codon 2921 8 bp target site duplication resulted in the insertion of an transcriptionally active Alu element

Stop at codon 2958

Stop at codon 2842

Non-alu/non-Alu NHE. 5’ BP Not stated, but appears as if it is located within exon 20 will result in an in-frame deletion of the last 34 aa of exon 20

Alu/Alu HR

Deletion, with Alu/non-Alu NHE size reported as 1518 bp, but according to the NG_012772.1 seq, size of del should be 1501 bp

4 953 bp deletion

Stop at codon 2525

Predicted effecte

Non-Alu/non-Alu HR. BPs Exons 17–19 are removed from 1 arise over a 5 nt repeat the transcript. Final result is a situated at both ends of the frameshift with termination at joined sequences codon 2635

Alu/Alu HR

Type of sequence at breakpoint & mechanism of rearrangementd

[51]

[13]

[7]

[56]

[13]

[7]

[83]

Reference

HGVS nomenclature—reported breakpoints, which are shown undera, have been converted, using the RefSeqGene record (NG_012772.1) and the recommended HGVS nomenclature

Type of sequence at breakpoint and mechanism of rearrangement: HR homologous recombination, NHE non-homologous event

Effect particular rearrangements is predicted to have on the transcript or protein. If cDNA analysis was not completed by the authors, the effect of the LGR is predicted using the NM_000059.3 sequence

Number of families identified with a particular rearrangement

? No. of families identified with this LGR was not indicated

Methods used to detect rearrangement: LR-PCR long-range PCR, MLPA multiplex ligation-dependent probe amplification, QMPSF quantitative multiplex PCR of short fluorescent fragments, SSCP single-strand conformation polymorphism analysis

g

f

aa amino acids

e

d

Size of LGR—the size indicated in this column may be different to that reported, since there may be differences in the reference sequences used. Please note that in this column the RefSeqGene record, NG_012772.1 was used to calculate these values

c

[Correct HGVS nomenclature cannot be given, since the seq of the inserted segment must be indicated. See discussion on nomenclature in text]

NG_012772.1:g.68883_68884insAlu

NG_012772.1:g.60200_65152del

1,155 bp deletion


10,842 bp deletion

2,463 bp deletion

Size of LGRc

Clone sequences utilized for identification of BRCA2 breakpoints are: AY436640, U43746 and the UCSC sequence [obtained from the Genomic locale from the Human Genome Browser, May 2004 assembly (http://genome.ucsc.edu/)]

b

a

NC_000013.9: g.55520_56675del1156

20

NG_012772.1:g.60519_61673del

Duplication with BPs at NG_012772.1:g.56115_65818dup 13:31838735 and 13:31848434 (UCSC sequence)

NG_012772.1: g.48887_59728del

NG_012772.1:g.45679_48141del

HGVS nomenclatureb

19–20

x


Exons

Table 2 continued



Furthermore, the standard lists (Tables 1, 2) of LGRs should also assist researchers in determining whether LGRs detected are novel or have previously been reported. Please note that the breakpoints of the LGRs listed in Tables 1 and 2 could only be verified in instances where electropherograms or sequences were provided by authors.

section originates from a sequence with a different accession number, the accession (and version) number and the exact nucleotides that are inserted should be indicated. If the large insertion originates from the same sequence (sequence with the same accession number), just the nucleotides involved need to be mentioned, for examples refer to the LGRs involving exons 21–22 (NG_005905.2:g.166375_170153 delins:g.162086_162321) described by Vasikova et al. [47] and Zikan et al. [48], as well as the LGR affecting exons 18–19 (NG_005905.2:g.152856_159830delins:g.33163_ 33189) reported by Engert et al. [30] in Table 1. Unfortunately, two LGRs involving both a deletion and an insertion (involving exons 15–20 of BRCA1, and exons 1–2 of BRCA2) and three large insertions (involving exon 11 of BRCA1; and exons 3 and 22 of BRCA2) could not be named fully according to the HGVS nomenclature because the sequence of the insertion was unknown/not given [10, 13, 34, 49–51]. Finally, duplications should be assigned by indicating the first and last nucleotides duplicated, followed by ‘‘dup’’. In the case of triplications and quadruplications, it is suggested to no longer use ‘‘trip’’ and ‘‘quad’’, but rather [3] and [4], and the nucleotide numbers of the section triplicated or duplicated should be indicated [27]. Please refer to the HGVS nomenclature for the exons 17–19 triplication (NG_005905.2:g.147982_156332 [3]) reported by Hogervorst et al. [18] in Table 1. We trust that these suggestions will make the description of BRCA1/2 LGRs less complex, and allow the manner in which it is done to be uniform amongst all groups.

A

gaggccaggc gaagctcagg 15931 actgcccctg 15991 tttttgtttt 16051 TCTCCAAACA 15811 15871

atctcatctt ggtacaaggc ttaaggtagg cctcccgcct GTTATACTGa

TCTCCAAACA acccttttta ttaaggtagc tggccgtgta gtatttggcg

B

Review of previously reported BRCA LGRs with mapped breakpoints For the remaining section of this article, only BRCA1/2 LGRs that were actually characterized are discussed. Please note that many more rearrangements that have not been characterized have been reported. The number of LGRs reported in BRCA1/2 has increased dramatically over the last 4 years. In the review by Mazoyer, published in 2005 [52], only 29 BRCA1 and three BRCA2 LGRs had been characterized. Only 4 years later, these values have almost tripled (81) for BRCA1 and almost sextupled (17) for BRCA2. This is mostly testimony to the increased use of MLPA. In fact, 49 of the 52 newly characterized BRCA1 LGRs have been detected using MLPA, and 9 of the 14 newly characterized BRCA2 LGRs. In total, MLPA has been responsible for the detection of 67 of the 81 (82.7%) BRCA1 LGRs, and 9 of the 17 BRCA2 LGRs (53%). These figures indicate how important this methodology has become since its development in 2001, and the significant role it has played in helping identify LGRs. GTTATACTGa gtagagatga tctaaataac cacctgtaag tccatcatca

gtagagatga ggtttcacct tttgatggtc ggtacaagga atttatattc

aagtgggtgt gataggcaag tgtgtatttg gttaggatcc tctgttaact

g.16070

g.15849

g.15850

C 15811 15871 15931 15991 16051

gaggccaggc gaagctcagg actgcccctg tttttgtttt TCTCCAAACA

atctcatctt ggtacaaggc ttaaggtagg cctcccgcct GTTATACTGa

TCTCCAAACA acccttttta ttaaggtagc tggccgtgta gtatttggcg

GTTATACTGa gtagagatga tctaaataac cacctgtaag tccatcatca

gtagagatga ggtttcacct tttgatggtc ggtacaagga atttatattc

aagtgggtgt gataggcaag tgtgtatttg gttaggatcc tctgttaact

g.16069

Fig. 5 Designation of breakpoints of large deletions A Reference Genomic sequence (L13789.4) of exon 4 and surrounding intronic regions of HYPO1 (Hypothetical gene 1) in which a deletion has been identified. Uppercase underlined sequence represents two regions of 19 bp that share 100% homology, which mediated the deletion. B Electropherogram

of the sequence of the deletion allele. Arrows indicate the nucleotides flanking the deleted sequence. Block shows the remaining homologous region. C Deleted sequence is shown in the box. The first deleted nucleotide is g.15850 and the last deleted nucleotide is g.16069. Deletion should therefore be named L13789.4:g.15850_16069del

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Breast Cancer Res Treat Table 3 Features of BRCA1/2 rearrangements

BRCA1 Total number LGRs characterized

BRCA2

81

Deletions

79% (64/81)

17 52.94% (9/17)

Insertions

1.23% (1/81)

11.76% (2/17)

Duplication

8.64% (7/81)

11.76% (2/17)

9.9% (8/81)

23.53% (4/17)

1.23% (1/81)

–

55.56% (45/81)

70.6% (12/17)

Deletion and insertion Triplication LGRs result in Premature truncation In-frame deletion

21% (17/81)

In-frame duplication

1.23% (1/81)

In-frame triplication Loss of transcript

5.88%(1/17)

1.23% (1/81)

–

14.81% (12/81)

5.88%(1/17)

Transcription of chimeric gene

4.94% (4/81)

–

Effect unknown (size of Alu insertion could not be determined)

1.23% (1/81)

–

Table 4 Mechanisms of BRCA1/2 rearrangements

BRCA1 Recombination between BRCA1 and wBRCA1 Alu-mediated unequal homologous recombination Non-homologous events

6.17% (5/81) 72.84% (59/81) 19.75 (16/81)

BRCA2 52.94% (9/17) 41.18% (7/17)

Alu/non-Alu

9.88% (8/81)

17.65% (3/17)

Non-Alu/non-Alu

9.88% (8/81)

23.53% (4/17)

1.23% (1/81)

5.88% (1/17)

Homologous recombination (no Alu Involved)

Recurrent and founder LGRs Of the 81 BRCA1 LGRs reported, 28 (34.6%) have been described more than once. Nine of these 28 recurrent LGRs have been shown to share a common haplotype. Perhaps, the most striking of these founders is the exon 13 duplication (NG_005905.1:g.133622_139702dup), which has been detected in at least 91 families. This founder has been identified in a variety of population groups, but appears to originate from the York and Trent regions in the UK [21, 52–55]. Four founder mutations have been detected in the Dutch population, two of which [DelEx13 (NG_005905.2:g. 133766_137600del) and DelEx22 (NG_005905.2:g.168 752_169261del)] account for 23% of all Dutch BRCA1 mutations [18]. Very recently, two different deletions of exons 1 and 2 (NG_005905.2:g.86268_94435delinsAA AAAAAAA and NG_005905.2:g.61101_98034del) have been shown to represent Dutch founders [40]. A deletion of exons 3–16 (NG_005905.2:g.101213_147798del) has been detected in 11 Danish families [46, 56]; although, both papers report the LGR using different reference sequences, the breakpoints according to the provided electropherograms, are identical. Hansen et al. [56] showed that the nine families positive for the LGR shared a common haplotype, indicating the presence of a founder LGR in the Danish

123

17.65 (3/17)

population. Other founder LGRs identified, include DelEx8– 9 (NG_005905.2:g.114552_121641del) in the American population [57], DelEx8–13 (NG_005905.2:g.116216_ 139982del) in the French [58, 59], DelEx9-12 (NG_00 5905.2:g.118955_133611del) in the Hispanic [41], InsEx11 (NG_005905.2:g.124192_124193insAlu) in the Belgian [50], and DelEx17 (NG_005905.2:g.148011_151126del) in the German/Italian populations [30, 32, 60]. Only one BRCA2 LGR founder mutation exists. This is the insertion of an Alu element in exon 3, and has been identified in at least 32 Portuguese families [10, 34, 50], and reportedly accounts for more than a quarter of deleterious BRCA mutations in Central/Northern Portugal [34].

Features of BRCA1/2 rearrangements To date, 81 distinct BRCA1 LGRs have been characterized (Table 1), and identified at least 291 times. The majority of BRCA1 LGRs (Table 3) are deletions (79%—64/81), while only one insertion and one triplication have been characterized. Seven duplications have been distinguished, and the remaining eight LGRs include both an insertion and a deletion. The size of the BRCA1 LGRs vary from just a few hundred base pairs, to tens of kilobases, with the smallest size


being 244 bp, deleting exon 5 (NG_005905.2:g.111421_ 111664del) and the largest 160,880 bp, (NG_005905.2:g. 8836_169713del), which removes NBR2, 18 of the 19 NBR1 exons and exons 1–22 of BRCA1. Fifty-six percent of the LGRs (45/81) result in premature termination of the BRCA1 protein, 21% (17/81) cause an in-frame deletion, 14.81% (12/81) prevent transcription, and 4.94% (4/81) result in the transcription of a chimeric gene that comprises exons 1A-2 of wBRCA1 fused to exons 3–24 of BRCA1. One of the LGRs results in an in-frame triplication of exons 17–19, one in an in-frame duplication of exons 18 and 19, and the effect of one LGR (NG_005905.2:g.124192_124193insAlu in exon 11) is unknown. Although the authors investigated the effect of the insertion on mRNA level, their results were inconclusive [50]. Also the exact size of the Alu insertion could not be determined and therefore one cannot predict what effect this insertion may have. Each exon of the BRCA1 gene has been involved in an LGR, but exon 24, the most terminal exon, has been involved the least. Although exon 20 has often been involved in multi-exon LGRs, it has been involved in an LGR on its own at least seven different times and may therefore represent a breakpoint hotspot. Interestingly, six different exon 1–2 deletions, with different breakpoints, have been reported 16 times in a number of different populations. Only the two reported by van den Ouweland et al. [40] appear to be founders. It is of particular interest that three of these six deletions are identical in size (36 934 bp), but have different breakpoints. The occurrence of this hot spot for recombination is explained by the presence of wBRCA1, 28-kb upstream from BRCA1, with which it shares homology. Alu-mediated unequal homologous recombination, is by far the most common event resulting in BRCA1 LGRs (Table 4), with as many as 59 of the 81 (72.84%) BRCA1 LGRs being mediated in this manner. Five of the 81 (6.17%) occurred as a result of recombination between BRCA1 and wBRCA1, and 19.75% (16/81) were caused by non-homologous events. Of the 16 nonhomologous events, 50% involve one Alu repeat, at either the 50 or 30 breakpoint. Interestingly, one of the LGRs (a deletion of exon 5) occurs as a result of homologous recombination, but without the involvement of an Alu repeat (NG_005905.2:g.111421_111664del) [30, 45]. Far fewer BRCA2 LGRs (17) have been characterized (Table 2) in comparison to BRCA1 (81). The 17 BRCA2 LGRs have been reported in 48 families. Of the characterized BRCA2 LGRs, 52.94% are deletions (9/17), 11.76% represent insertions (2/17), 11.76% duplications (2/17), and 23.53% (4/17) a simultaneous deletion and insertion events (Table 3). Approximately 71% (12/17) of the LGRs result in premature truncation of the protein, 18% (3/17) cause an inframe deletion and 6% (1/17) an in-frame duplication, and the remaining 6% (1/17) loss of transcription of the deletion

allele (Table 3). The five most terminal exons of BRCA2 have not been involved in an LGR. The smallest BRCA2 LGRs are the Alu insertions of *300 bp in exons 3 and 22 (NG_012772.1:g.8686_8687insAluS; NG_012772.1:g.68883_ 68884insAlu), while the largest is the 16,175 bp duplication from exon 4 up to and including 56% of exon 11 (NG_012772.1:g.16099_30416del). Alu-mediated unequal homologous recombination is responsible for 52.94% (9/17) of BRCA2 LGRs (Table 4). Seven of the seventeen LGRs (41.18%) were caused by non-homologous events, three of which involve one Alu repeat, at either the 50 or 30 breakpoint. Interestingly, as with BRCA1, one of the LGRs (deletion of exons 17–18) occurs as a result of homologous recombination, but without the involvement of an Alu repeat (NG_012772.1:g.48887_59728del) [7]. Although both BRCA1 and BRCA2 contain particularly high densities of repetitive elements (47% each), BRCA1 contains more than double the amount of Alu elements than BRCA2 (42 vs. 20%) [6, 61]. It is therefore not surprising that Alu-mediated unequal homologous recombination is more common in BRCA1 than BRCA2 LGRs (72.8% in BRCA1 vs. 52.9% in BRCA2; Table 4), while non-homologous events are more common in BRCA2 LGRs (Table 4). Deletions are far more common in BRCA1 than BRCA2, while insertions and particularly insertion–deletions occur more frequently in BRCA2 (Table 3). Also of interest, is the fact that BRCA2 LGRs result in premature truncation of the protein more often than BRCA1 LGRs (70.6% vs. 55.6%). Although, BRCA2 contains approximately half the amount of Alu repeats than BRCA1, it is by no means a small amount. The smaller numbers of reports of BRCA2 LGRs is often attributed to the fact that BRCA2 contains fewer Alus than BRCA1, but could this not also be a function of the fact that initial reports indicated that BRCA2 LGRs were absent/uncommon, causing most researches to focus their attention on BRCA1, and therefore, to some extent neglecting BRCA2? Of course, these values may also be accurate reflections, and BRCA2 may just not be involved that frequently. In conclusion, we have detected a novel BRCA1 deletion of 11,053 bp, removing exons 23–24 (NG_005905.2:g. 169527_180579del) in a South African HBC patient. The identification of this single LGR in the South African population, and the absence of identification of any LGRs in the Afrikaner population indicates that MLPA should not be used as an initial screen for South African/Afrikaner HBC as is suggested in other populations [8, 22]. Furthermore, we have converted the breakpoints of all characterized BRCA1/2 LGRs published to date, to the RefSeqGene records, NG_005905.2 or NG_012772.1, using the recommended HGVS nomenclature. We have therefore now created a standardized/uniform resource which will assist researchers in determining whether ‘‘new’’ LGRs are actually novel.

123


Furthermore, we attempted to clarify the HGVS rules of nomenclature, which appear to have been frequently misunderstood in the past. We hope that the tools provided here will make standardized reporting of BRCA1/2 LGRs easier in the future. Acknowledgments We thank Gerard Pals for assistance with the MLPA technology and supplying us with positive controls. Also, our sincere thanks to Jan Schouten for endorsing us with MLPA kits, and Johan den Dunnen for assistance with the nomenclature. This study was supported by a research grant from the Cancer Association of South Africa (CANSA) to E. J. van Rensburg.

10.

11.

12.

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