MATERIAL AND METHODS

Patients

A total of 101 unrelated patients affected by colorectal cancer or an HNPCC associated cancer (endometrium, small bowel, urinary tract) were from 17 families which fulfilled the Amsterdam II criteria¹² and from 84 families matching our modified criteria of suspected HNPCC, one colorectal cancer patient with a first degree relative affected by an HNPCC associated cancer, one of whom was diagnosed under the age of 50 years.¹³ The clinical diagnosis of HNPCC was established or verified at the Hereditary Cancer Centre, Pomeranian Academy of Medicine, Szczecin, Poland. Patients used for this study were ascertained from the following regions: Bydgoszcz (3), Gdańsk (3), Kielce (14), Kraków (3), Legnica (1), Lublin (2), Lódz (1), Olsztyn (13), Poznań (7), Riga (3), Szczecin (33), Tartu (3), Wrocław (5), Vilnius (6), Zielona Góra (4).

DNA isolation

Peripheral blood samples were collected from the patients after obtaining informed consent. DNA was extracted directly from leucocytes by the classical phenol purification method or as described previously.¹⁴

DNA sequencing

All exons and exon-intron junctions of MLH1 and MSH2 were amplified using the same protocols as described previously¹⁵ with the same primer sequences as described by Wijnen et al^16,17 for DGGE but without the M13 and GC clamp sequences at the 5‘ end. Dye terminator cycle sequencing reactions were performed using the ABI PRISM Dye-terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer) according to the manufacturer’s recommended protocol. Semi-automated fluorescence analysis was performed using a 373 A DNA Sequencer (ABI, Perkin-Elmer).

RNA based sequencing

RNA sequencing template was used for 15 patients in whom mutations had not previously been detected by genomic DNA based sequencing. Total RNA was extracted from fresh lymphocytes using Trizol (Gibko BRL, Life Technologies Inc, Gaithersburg, MD). cDNA was synthesised with c therm polymerase (Roche Diagnostics) using 0.5 μg total RNA and specific primers complementary to the 3‘ end of MLH1 and to the 3‘ end of MSH2. PCR products were size fractionated by agarose gel electrophoresis. Sequencing of the cDNA was as described previously.¹⁸

RESULTS

Unequivocal mutations were detected in 34 families; 16 mutations were identified in MSH2 and 18 mutations in MLH1. Among families which fulfilled the Amsterdam II criteria mutations were identified in 59% of kindreds. There were three families that harboured MSH2 changes and seven families associated with MLH1 gene mutations. From the patients matching our modified criteria of suspected HNPCC, mutations were detected in 29% of families, 13 in MSH2 and 11 in MLH1.

Recurrent mutations were identified in 17 (50%) families with mutations. Two of the most frequent changes were a substitution of A to T in the splice donor site of intron 5 of the MSH2 gene, which represents the most frequent MSH2 mutation reported⁸ and a missense change, a A681T in MLH1, found in seven and six families, respectively. Mutations not described previously in other populations were found in nine families (highlighted in table 1, fig 1). The pathogenic nature of the g to c change at 1661+5 was confirmed at the RNA level by identifying an aberrant transcript which was the result of exon 10 skipping which resulted in the creation of stop codon. RNA based sequencing resulted in the detection of an aberrant MLH1 transcript which was the result of a loss of exon 16, but no change could be identified in genomic DNA.¹⁸ In addition to unequivocal mutations, sequence variants of uncertain pathological significance were detected in five families. One of these alterations (c.875T>C) occurred in two patients (table 1). Frequent and rare MSH2 and MLH1 polymorphisms are summarised in table 2.

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Figure 1

MLH1 and MSH2 mutations not described in other populations. Sequence chromatograms of cases 2, 12, 13, 15, 16, 17, 22, 24, and 27.

A summary of novel missense alterations of unknown significance is shown in table 3.

DISCUSSION

The appropriate management of patients from HNPCC families decreases the risk of cancer and results in a better life expectancy. The identification of HNPCC families remains problematical, as the Amsterdam II criteria which rely on pedigree and clinical data are very restrictive. Indeed the above Amsterdam II criteria cannot be matched in the majority of families with MSH2 or MLH1 mutations. At present, the only method to diagnose HNPCC unequivocally in suspected cases of this disorder is to identify constitutional mutations in the genes associated with this disorder. The most accurate method of detecting germline mutations are molecular analyses using either DNA or RNA templates for DNA sequencing analysis. These techniques are complex, time consuming, and expensive. Given the limited resources for routine mutation screening, it is essential to develop DNA testing protocols further. One of the most efficient ways of achieving this is to describe the nature and frequency of population specific mutations in order to target those changes first. From the results presented here, it is apparent that two mutations (a to t substitution in the splice donor site of intron 5 of the MSH2 gene and missense A681T in MLH1) account for about 40% (12/34) of mutations identified in Polish HNPCC families. Two other mutations (1216C>T in MSH2 and 83C>T in MLH1) occurred in two families (one of which has not previously been reported) and eight mutations (del273TCT, 2131C>T in MSH2 and 184C>T, 350C>T, 667G>T, g>c at 1409+1, ins C at 1490, and 1672G>T in MLH1) were observed previously in other populations. The remaining nine mutations (613G>T, g>c at 1661+5, ins A at 1772, g>c at 2210+1, delT at 2388 in MSH2 and delG at 37, ins AA at 356, delAGgt at 883, delCCA at 1658 in MLH1) were found to be potentially specific for Polish families as they have not previously been described. Therefore, the characteristics of mutations identified from Poland and the Baltic States suggest that it is worthwhile to develop mutation testing focusing on these changes as a first approach. The frequency of mutations in families that fulfilled the Amsterdam II criteria was approximately 60% and in families suspected of HNPCC around 30%, which is comparable with the frequency of mutations identified by other authors for different populations.¹⁹ Therefore, it can be predicted that the sensitivity of testing of this population for the reported mutations could be very high. Certainly, the sensitivity of this testing approach will not achieve 100%. False negative results could potentially occur in families with missense mutations. In our series, more than 10% of MSH2/MLH1 mutations involved such an amino acid substitution (table 3). The missense alteration 2558A→C at codon 853 in exon 15, which resulted in a substitution of glutamic acid for alanine, seems to be a neutral rare polymorphism as it did not cosegregate with the disease in the family. In spite of the location in important functional domains in the MLH1 protein, two missense alterations, I19F and L292P, could not be exclusively associated with the disease as they may represent polymorphisms. The missense alteration 2059C→T at codon 687 in exon 18 that resulted in substitution of arginine for tryptophan appears to be a pathogenic alteration. The sensitivity of finding MSH2/MLH1 mutations can be further improved by the detection of large genomic deletions or rearrangements. Their contribution may be as high as 36% of all MSH2 mutations according to Dutch HNPCC mutation analysis.²⁰MLH1 mutations are mainly single nucleotide changes. The only country with a high proportion of MLH1 deletion is Finland, where a founder effect associated with a 3.5 kb deletion encompassing exon 16 has been identified.¹⁰ Our studies of RNA transcripts suggest that the frequency of large intragenic deletions without involvement of the 5‘ and 3‘ ends of the MSH2 or MLH1 genes is low in the population studied here. However, more extensive studies are necessary in order to describe the actual proportion of such changes among HNPCC families in this population. Loss of exon 16 detected at the RNA level may suggest the presence of a large genomic deletion. This is not, however, caused by the 3.5 kb genomic deletion observed frequently in Finland, since in experiments using long PCR with primers for exons 15 and 17 we observed only the product of normal length.¹⁸ Two of the most frequent mutations identified in Poland were also found in Lithuanian families, suggesting a common history. Poland and the Baltic States may have more common mutations than reported here since the number of samples from Estonia, Latvia, and Lithuania were too small to make the appropriate comparisons. In summary, it seems likely that the MSH2 and MLH1 changes described here are representative of the majority of HNPCC mutations in families from this region. Therefore, we believe it is justified to develop a DNA testing strategy based on the preferential analysis of changes identified from this population.