High proportion of large genomic rearrangements in hMSH2 in hereditary nonpolyposis colorectal cancer (HNPCC) families of the Basque Country
Introduction
Hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) is an autosomal dominant disorder which is estimated to account for al least 5% of all cases of colorectal cancer (CRC); in the Spanish population, it appears to be responsible for 2.5% of all cases of CRC [1]. HNPCC is characterised by cancer of the colon, rectum, endometrium, small bowel or urinary tract of early onset. In the majority of cases, Lynch syndrome is caused by germline mutations in the mismatch repair genes (MMR) MLH1, MSH2, MSH6, PMS2 or MLH3[2], with MSH2 and MLH1 being the most important [3]. The MMR genes code for molecules, which repair the errors in the DNA copy. Mutations in these genes inactive the DNA editing function.
Many of the germline mutations described are frameshift, nonsense, splicing or missense. Recently, development of the multiplex ligation-dependent probe amplification (MLPA) technique has enabled large genome rearrangements to be studied. In addition, MLPA enables a greater quantity of sample to be analysed in a shorter period of time, making it possible to observe that these genomic rearrangements account for a significant percentage of the mutations in MSH2 and MLH1[4].
A previous study of the Spanish population performed by Castellvi-Bell et al. [5] revealed a lower frequency of rearrangements in the MSH2 and MLH1 genes (<1.5%) than had been found in other European populations, with frequencies between 15% and 55% [6], [7]. However, few studies on HNPCC have used the MLPA technique. We have therefore undertaken this study on a Basque Country population in order to determine the frequency of large rearrangements in the population with HNPCC. A comparison of the frequency of anomalies was made with the European and Spanish population, as has previously been performed by our group for breast cancer [8].
Section snippets
Families
This study includes a total of 29 unrelated index cases corresponding to: (a) 19 index cases in unrelated families who satisfied Amsterdam criteria I or II for HNPCC; (b) 10 index cases in unrelated families who satisfied the revised criteria of the Bethesda guidelines [9]: and finally we also studied 29 relatives of the positive cases.
Molecular analysis
Informed consent was obtained prior to the extraction of peripheral blood samples for the genetic analyses. Our complete molecular analysis protocol comprises
Results
MLPA analysis of the genome rearrangements for the 29 index cases revealed six cases in which alterations were found, four in MSH2 gene, and two in MLH1 gene. All six positive cases met the Amsterdam criteria. The four rearrangements in MSH2 correspond to deletions, in which more than one exon is lost. The MLH1 rearrangements were initially detected as one deletion of exon 18 and one deletion of exon 19 but, after sequencing analysis, these deletions were not confirmed and correspond to base
Discussion
Although we initially appeared to have found six genomic rearrangements by means of MLPA, in reality there are only four, all of which are in MSH2. Of the alterations in MLH1, one was shown to be a deletion of two base pairs (Fig. 2), and the other, a missense mutation. Although both alterations are pathogenic, they do not constitute large rearrangements. This demonstrates the need to use other techniques to confirm alterations detected by MLPA, particularly in those cases in which the
Acknowledgement
This work was supported by a Grant from the Basque Government Department of Health (Grant No. 200411060).
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