Loss of heterozygosity on 10q and mutational status of PTEN and BMPR1A in colorectal primary tumours and metastases

Abstract

We investigated the possible role of chromosome 10q losses in colorectal cancer metastasis by carrying out an allelic imbalance study on a series of microsatellite instability-negative (MSI-) primary tumours (n=32) and metastases (n=36) from 49 patients. Our results demonstrate that 10q allelic losses are associated with a significant proportion (25%) of MSI- colorectal tumours, but are not involved in the metastatic process. PTEN and BMPR1A, two genes located in the common deleted region, were screened for mutations in samples with loss of heterozygosity. The absence or low frequency of mutations indicates that the inactivation of these genes by deletion of one allele and mutation of the other one plays only a minor role in MSI- tumours.

Figure 1

Deletion mapping of chromosome 10q. Allelic patterns of chromosome 10q for all tumour samples with LOH are shown. T: primary tumour; L: liver metastasis. Plain ovals: no loss of heterozygosity in the tumour sample; black ovals: loss of heterozygosity in the tumour sample; striped ovals: not informative (homozygosity in the normal sample); blank space: not done. Names of microsatellite markers studied, their positions on 10q and their genetic distance to the top of the chromosome are indicated on the left. The minimal region of loss and the location of the BMPR1A and PTEN genes are shown on the right.

Figure 2

PTEN mutation in the primary tumour and liver metastasis of case 26. (A) Abnormal bands were detected by SSCP analysis of cDNA from the primary tumour (T) and liver metastasis (M) using primers in exon 5 (sense) and exon 6 (antisense). These bands were not present in normal colon cDNA from the same patient (N). (B) Sequencing analysis of the two main abnormal bands (T2) and (T3) present in the primary tumour. Sequence of the normal cDNA from the same patient (N). (C) Sequencing of the genomic DNA of the primary tumour (T) and corresponding normal tissue (N). Tumour DNA harboured a G to T point mutation. (D) The various alternatively spliced forms deduced from the cDNA and genomic sequences presented in (B) and (C) are shown. The T2 allele carrying a G/T transversion in exon 5 presented the same splice form as the normal allele. T3 showed a 21 bp deletion at the 3′ end of exon 5. The new consensus donor splice site created by the mutation is underlined. (E) RT–PCR analysis of the primary tumour (T), liver metastasis (M) and corresponding normal tissue using the same primers as in (A). Lane 1: pBR322 DNA-MSPI digest.