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Germline CDH1 deletions in hereditary diffuse gastric cancer families.

Oliveira C, Senz J, Kaurah P, Pinheiro H, Sanges R, Haegert A, Corso G, Schouten J, Fitzgerald R, Vogelsang H, Keller G, Dwerryhouse S, Grimmer D, Chin SF, Yang HK, Jackson CE, Seruca R, Roviello F, Stupka E, Caldas C, Huntsman D - Hum. Mol. Genet. (2009)

Bottom Line: The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions.CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences.As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Pathology and Immunology, University of Porto, Portugal.

ABSTRACT
Germline CDH1 point or small frameshift mutations can be identified in 30-50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT-PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is approximately 46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

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Array CGH profiles obtained for HDGC families carrying large CDH1 deletions. Examples are shown for four of the six families. See Tables 2 and 3 for details on the deletion found in each family. Deletions are represented by dense areas of green dots on the left side of each panel. Vertical thick gray bars on the left represent genes in the genomic area surrounding the CDH1 locus and corresponding gene symbols are also shown. Smaller deletions in families#3 and #5 are pointed with black arrows. Please note the scale in Mb on the right, for chromosome position.
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DDP046F2: Array CGH profiles obtained for HDGC families carrying large CDH1 deletions. Examples are shown for four of the six families. See Tables 2 and 3 for details on the deletion found in each family. Deletions are represented by dense areas of green dots on the left side of each panel. Vertical thick gray bars on the left represent genes in the genomic area surrounding the CDH1 locus and corresponding gene symbols are also shown. Smaller deletions in families#3 and #5 are pointed with black arrows. Please note the scale in Mb on the right, for chromosome position.

Mentions: A combination of array CGH and PCR was utilized to map the putative breakpoints in five of the six probands (Fig. 2). The array CGH indicated the regions of chromosome 16 at which the breakpoints were located. PCR primers were then designed to span the breakpoints and the resulting PCR products sequenced (Table 2). Family 1 and family 2 showed identical MLPA patterns as well as virtually identical array CGH results. Sequencing of the breakpoint revealed that these two families harbored an identical 193 593 bp deletion encompassing the full sequence of CDH3 and extending to position IVS2+57 595 in CDH1. Family 3 was found to carry a 5671 bp deletion with breakpoints at position −3809 upstream of the CDH1 transcription start site (TSS) and at position IVS2+742 in CDH1. Family 4 harbored a 150 bp deletion encompassing the TSS of CDH1, with breakpoints located 125 bp upstream and 25 bp downstream of the TSS. Array CGH was not done for this proband since the PCR encompassing CDH1 exon 1 produced both a normal sized and a shorter band that was visible by dHPLC and further used to clone the breakpoints for the deletion by direct sequencing. Family 5 carried an 8078 bp deletion ranging from IVS13-2738 to 20 bp downstream of the stop codon of CDH1, as confirmed by long-range PCR using specific primers flanking the breakpoints (Fig. 3A). Family 6 was found to carry an 828 bp deletion ranging from IVS15+3097 to 223 bp downstream of the stop codon of CDH1 (Fig. 3B).


Germline CDH1 deletions in hereditary diffuse gastric cancer families.

Oliveira C, Senz J, Kaurah P, Pinheiro H, Sanges R, Haegert A, Corso G, Schouten J, Fitzgerald R, Vogelsang H, Keller G, Dwerryhouse S, Grimmer D, Chin SF, Yang HK, Jackson CE, Seruca R, Roviello F, Stupka E, Caldas C, Huntsman D - Hum. Mol. Genet. (2009)

Array CGH profiles obtained for HDGC families carrying large CDH1 deletions. Examples are shown for four of the six families. See Tables 2 and 3 for details on the deletion found in each family. Deletions are represented by dense areas of green dots on the left side of each panel. Vertical thick gray bars on the left represent genes in the genomic area surrounding the CDH1 locus and corresponding gene symbols are also shown. Smaller deletions in families#3 and #5 are pointed with black arrows. Please note the scale in Mb on the right, for chromosome position.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667284&req=5

DDP046F2: Array CGH profiles obtained for HDGC families carrying large CDH1 deletions. Examples are shown for four of the six families. See Tables 2 and 3 for details on the deletion found in each family. Deletions are represented by dense areas of green dots on the left side of each panel. Vertical thick gray bars on the left represent genes in the genomic area surrounding the CDH1 locus and corresponding gene symbols are also shown. Smaller deletions in families#3 and #5 are pointed with black arrows. Please note the scale in Mb on the right, for chromosome position.
Mentions: A combination of array CGH and PCR was utilized to map the putative breakpoints in five of the six probands (Fig. 2). The array CGH indicated the regions of chromosome 16 at which the breakpoints were located. PCR primers were then designed to span the breakpoints and the resulting PCR products sequenced (Table 2). Family 1 and family 2 showed identical MLPA patterns as well as virtually identical array CGH results. Sequencing of the breakpoint revealed that these two families harbored an identical 193 593 bp deletion encompassing the full sequence of CDH3 and extending to position IVS2+57 595 in CDH1. Family 3 was found to carry a 5671 bp deletion with breakpoints at position −3809 upstream of the CDH1 transcription start site (TSS) and at position IVS2+742 in CDH1. Family 4 harbored a 150 bp deletion encompassing the TSS of CDH1, with breakpoints located 125 bp upstream and 25 bp downstream of the TSS. Array CGH was not done for this proband since the PCR encompassing CDH1 exon 1 produced both a normal sized and a shorter band that was visible by dHPLC and further used to clone the breakpoints for the deletion by direct sequencing. Family 5 carried an 8078 bp deletion ranging from IVS13-2738 to 20 bp downstream of the stop codon of CDH1, as confirmed by long-range PCR using specific primers flanking the breakpoints (Fig. 3A). Family 6 was found to carry an 828 bp deletion ranging from IVS15+3097 to 223 bp downstream of the stop codon of CDH1 (Fig. 3B).

Bottom Line: The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions.CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences.As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Pathology and Immunology, University of Porto, Portugal.

ABSTRACT
Germline CDH1 point or small frameshift mutations can be identified in 30-50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT-PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is approximately 46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

Show MeSH
Related in: MedlinePlus