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Medium-sized deletion in the BRCA1 gene: Limitations of Sanger sequencing and MLPA analyses.

Herman S, Varga D, Deissler HL, Kreienberg R, Deissler H - Genet. Mol. Biol. (2012)

Bottom Line: Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion.We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions.Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynaecology, University of Ulm, Ulm, Germany.

ABSTRACT
We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification. This showed that a considerable fraction of medium-sized deletions are undetectable by currently used standard methods of mutation analyses. We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions. Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.

No MeSH data available.


Related in: MedlinePlus

Detection of a medium-sized deletion in the BRCA1 gene. A: Family with a history of breast and ovarian cancer in which a deletion in BRCA1 was found. Two sisters of a woman who had developed breast cancer at age 30 harboured this mutation (M), indicated are manifestations of breast cancer (BC), ovarian cancer (OC) and colon cancer (CC). B: Characterization of the 374 bp deletion in exon 11 by long PCR. Amplified genomic regions included exon 10 (E10) and the following intron and parts of exon 11 (E11), as shown in the top scheme. The deletion in one allele resulted in double bands of longer PCR products but not of the smallest one, which was generated with the reverse primer also used in routine sequencing. The amplicon used for sequence analysis is shown in the scheme above the products of long PCR (A-E). Marker fragments labelled with an asterisk are 1500 bp, 3000 bp and 6000 bp.
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f1-gmb-35-1-53: Detection of a medium-sized deletion in the BRCA1 gene. A: Family with a history of breast and ovarian cancer in which a deletion in BRCA1 was found. Two sisters of a woman who had developed breast cancer at age 30 harboured this mutation (M), indicated are manifestations of breast cancer (BC), ovarian cancer (OC) and colon cancer (CC). B: Characterization of the 374 bp deletion in exon 11 by long PCR. Amplified genomic regions included exon 10 (E10) and the following intron and parts of exon 11 (E11), as shown in the top scheme. The deletion in one allele resulted in double bands of longer PCR products but not of the smallest one, which was generated with the reverse primer also used in routine sequencing. The amplicon used for sequence analysis is shown in the scheme above the products of long PCR (A-E). Marker fragments labelled with an asterisk are 1500 bp, 3000 bp and 6000 bp.

Mentions: Genetic counselling and subsequent analyses of predisposing genes was requested by a 53 years old woman on the basis of her sister’s early-onset breast cancer and other cases of breast and ovarian cancer in her family (Figure 1A). Accordingly, her risk of developing breast cancer was calculated to be 31%. By sequencing of all exons and adjacent regions of the BRCA1 and BRCA2 genes, no pathogenic mutation was detected. Subsequent MLPA analyses showed a substantially reduced signal resulting from only one of two MLPA probes binding to BRCA1 exon 11, suggesting a deletion including a region close to the beginning of this exon. Long PCR with primers from the end of exon 10 to various positions of exon 11 confirmed a heterozygous medium-sized deletion (Figure 1B). These PCR products were subjected to sequence analysis which showed a deletion (del c.671-26_1018) of 374 bp. This deletion affected the binding site of one of the primers used to amplify the DNA prior to sequencing for which, as a consequence, only PCR products from the normal allele were obtained. This case raised the question as to the likeliness of detecting such medium-sized deletions by routine methodology.


Medium-sized deletion in the BRCA1 gene: Limitations of Sanger sequencing and MLPA analyses.

Herman S, Varga D, Deissler HL, Kreienberg R, Deissler H - Genet. Mol. Biol. (2012)

Detection of a medium-sized deletion in the BRCA1 gene. A: Family with a history of breast and ovarian cancer in which a deletion in BRCA1 was found. Two sisters of a woman who had developed breast cancer at age 30 harboured this mutation (M), indicated are manifestations of breast cancer (BC), ovarian cancer (OC) and colon cancer (CC). B: Characterization of the 374 bp deletion in exon 11 by long PCR. Amplified genomic regions included exon 10 (E10) and the following intron and parts of exon 11 (E11), as shown in the top scheme. The deletion in one allele resulted in double bands of longer PCR products but not of the smallest one, which was generated with the reverse primer also used in routine sequencing. The amplicon used for sequence analysis is shown in the scheme above the products of long PCR (A-E). Marker fragments labelled with an asterisk are 1500 bp, 3000 bp and 6000 bp.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3313516&req=5

f1-gmb-35-1-53: Detection of a medium-sized deletion in the BRCA1 gene. A: Family with a history of breast and ovarian cancer in which a deletion in BRCA1 was found. Two sisters of a woman who had developed breast cancer at age 30 harboured this mutation (M), indicated are manifestations of breast cancer (BC), ovarian cancer (OC) and colon cancer (CC). B: Characterization of the 374 bp deletion in exon 11 by long PCR. Amplified genomic regions included exon 10 (E10) and the following intron and parts of exon 11 (E11), as shown in the top scheme. The deletion in one allele resulted in double bands of longer PCR products but not of the smallest one, which was generated with the reverse primer also used in routine sequencing. The amplicon used for sequence analysis is shown in the scheme above the products of long PCR (A-E). Marker fragments labelled with an asterisk are 1500 bp, 3000 bp and 6000 bp.
Mentions: Genetic counselling and subsequent analyses of predisposing genes was requested by a 53 years old woman on the basis of her sister’s early-onset breast cancer and other cases of breast and ovarian cancer in her family (Figure 1A). Accordingly, her risk of developing breast cancer was calculated to be 31%. By sequencing of all exons and adjacent regions of the BRCA1 and BRCA2 genes, no pathogenic mutation was detected. Subsequent MLPA analyses showed a substantially reduced signal resulting from only one of two MLPA probes binding to BRCA1 exon 11, suggesting a deletion including a region close to the beginning of this exon. Long PCR with primers from the end of exon 10 to various positions of exon 11 confirmed a heterozygous medium-sized deletion (Figure 1B). These PCR products were subjected to sequence analysis which showed a deletion (del c.671-26_1018) of 374 bp. This deletion affected the binding site of one of the primers used to amplify the DNA prior to sequencing for which, as a consequence, only PCR products from the normal allele were obtained. This case raised the question as to the likeliness of detecting such medium-sized deletions by routine methodology.

Bottom Line: Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion.We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions.Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynaecology, University of Ulm, Ulm, Germany.

ABSTRACT
We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification. This showed that a considerable fraction of medium-sized deletions are undetectable by currently used standard methods of mutation analyses. We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions. Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.

No MeSH data available.


Related in: MedlinePlus