Germline CDH1 deletions in hereditary diffuse gastric cancer families

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.

Figure 1.

MLPA output from the six HDGC families carrying germline deletions of the CDH1 locus. The first 17 bars represent signal obtained with MLPA probes within the CDH1 gene. Please note that for exon 1, three probes were used (1a, 1b and 1c). CDH1 deletions are represented, in the graph, by smaller bars marked with arrows. Bars on the right hand-side represent results obtained with control probes for MLPA (n = 15).

Figure 2.

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.

Figure 3.

Sequencing chromatogram of the mapped breakpoints in family 5 (A) and family 6 (B). Comparison with the human genome reference sequence. (A) Deletion of 8078 bp without insertion of P or N nucleotides. (B) Deletion of 828 bp and insertion of a triplet at the breakpoint.

Figure 4.

Analysis of sequences flanking the deletion breakpoints. (A and B) Homology analysis of AluS type elements flanking deletions in families 1, 2 and 3. (C) Ensembl based scheme displaying the repetitive elements flanking the deletion in family 5.

Figure 5.

Haplotype analysis of family 1 and family 2 probands. (A) Scheme of chromosome 16 with reference to microsatellite markers used in the haplotype analysis and to the breakpoints of the deletion. (B) Common haplotypes displayed by probands of family 1 and family 2 and proving a common ancestry. (C) Agarose gel of the PCR that allowed the mapping of the deletion, showing similar band sizes for probands in both families.

Figure 6.

Frequency of CDH1 alterations in a series of 160 HDGC families, subdivided by GC incidence rates as well as by type of gene alteration (point or small frameshift mutations and large deletions).