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Human Molecular Genetics Advance Access originally published online on November 24, 2004
Human Molecular Genetics 2005 14(2):235-239; doi:10.1093/hmg/ddi021
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Human Molecular Genetics, Vol. 14, No. 2 © Oxford University Press 2005; all rights reserved

Low levels of microsatellite instability characterize MLH1 and MSH2 HNPCC carriers before tumor diagnosis

Hafid Alazzouzi1, Enric Domingo1, Sara González2, Ignacio Blanco3, Manel Armengol1, Eloi Espín1, Alberto Plaja1, Simó Schwartz1, Gabriel Capella2 and Simó Schwartz, Jr1,*

1Molecular Oncology and Aging Research, Centre d'Investigacions en Bioquímica i Biologia Molecular (CIBBIM), Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron 119-129, Barcelona 08035, Spain, 2Translational Research Unit and 3Genetic Counseling Unit, Institut Català d'Oncologia, Gran Via s/n Km 2.7, 08907 L'Hospitalet, Spain

* To whom correspondence should be addressed. Tel: +34 934894060; Fax: +34 934894040; Email: sschwartz{at}vhebron.net

Received July 28, 2004; Revised September 22, 2004; Accepted November 9, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Microsatellite instability (MSI) characterizes tumors arising in patients with hereditary non-polyposis colorectal cancer (HNPCC) syndrome. HNPCC is a hereditary autosomal dominant disease caused by germline mutations in genes from the DNA (MMR) mismatch repair system. In these tumors, the loss of MMR compromises the genome integrity, allowing the progressive accumulation of mutations and the establishment of a mutator phenotype in a recessive manner. It is not clear, however, whether MSI can be detected in HNPCC carriers before tumor diagnosis. The aim of this study was to evaluate the presence of genetic instability in MMR gene carriers in peripheral blood lymphocytes of carriers and non-carriers members of two HNPCC families harboring a germline MLH1 and MSH2 mutation, respectively. An extensive analysis of the allelic distribution of single molecules of the polyA tract bat26 was performed using a highly sensitive PCR-cloning approach. In non-carriers, the allelic distribution of single bat26 molecules followed a gaussian distribution with no bat26 alleles shorter than (A)21. All mutation carriers showed unstable alleles [(A)20 or shorter] with an overall frequency of 5.6% (102/1814). We therefore suggest that low levels of genomic instability characterize MMR mutation carriers. These observations suggest that somatic mutations accumulate well before tumor diagnosis. Even though it is not clear whether this is due to the presence of a small percentage of cells with lost MMR or due to MMR haploinsufficiency, detection of these short unstable alleles might help in the identification of asymptomatic carriers belonging to families with no detectable MMR gene mutations.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tumors from the hereditary non-polyposis colorectal cancer (HNPCC) syndrome are characterized by a widespread genomic instability phenotype, caused by a defective mismatch repair (MMR) system (1Go,2Go). Up to 50% of HNPCC patients carry germline heterozygous mutations in one of the DNA repair genes MLH1, MSH2 and MSH6 (2Go–4Go). In these families, non-neoplastic cells from HNPCC carriers are phenotypically normal and retain a wild-type allele of the MMR gene. The inactivation or loss of the wild-type allele results in the loss of the MMR function and causes, in HNPCC tumors, a mutator phenotype in a recessive manner (5Go,6Go). The defective MMR system allows the accumulation in a stepwise model of thousands of mutations in microsatellites and other repetitive sequences, generating microsatellite instability (MSI) and initiating the tumorigenic process that leads to MSI colorectal cancer (7Go–9Go). Loss of MMR is an early event in HNPCC cancer development that occurs before gatekeeper mutations are selected (10Go–12Go).

In concordance with the recessive nature of MMR deficiency, microsatellite alterations are typically absent in non-neoplastic cells from HNPCC patients, suggesting the proficiency of the MMR system in these cells (13Go,14Go). Also, extensive somatic microsatellite mutations have been found in normal tissues from rare individuals with congenital bi-allelic or dominant-negative mutations in MMR genes (15Go–17Go). However, the acknowledged recessive nature of MMR deficiency has been challenged by the scarce evidences showing that normal non-neoplastic cells from HNPCC individuals may be also MMR-deficient (18Go,19Go).

One of the microsatellites used for detecting genomic instability is bat26, a quasi-monomorphic mononucleotide repeat of 26 adenines (20Go,21Go). Because of the inherent instability of microsatellite sequences, the analysis of a microsatellite sequence in bulk tissues often results in a polymorphic pattern of different lengths in a gaussian-like distribution. With the majority of the current analytical methods, only the most predominant alleles are detectable. Even though low levels of instability can generate shorter alleles at a low frequency, these might be under the sensitivity thresholds for detection. This dilution factor can be overcome by using alternative methods that allow the analysis of single microsatellite molecules. The aim of this study has been to investigate the presence of genetic instability in peripheral blood cells obtained from carrier and non-carrier members of two well-characterized HNPCC families harboring MLH1 and MSH2 germline mutations, respectively, in the absence of detectable neoplasia. Length distribution pattern of single molecules of bat26 has revealed the appearance of unstable alleles before tumor diagnosis in MMR mutation carriers. These observations suggest that the detection of these short unstable alleles might help in the identification of asymptomatic carriers belonging to families with no detectable MMR gene mutations and may be of help in further assessing the pathogenicity of missense MMR mutations.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Tumors from the HNPCC syndrome show high levels of MSI and a defective MMR cellular environment (1Go,2Go). In these tumors, the loss or inactivation of the remaining wild-type allele of the MMR gene is believed to cause the inactivation of the MMR system starting a tumorigenic mutator phenotype (8Go,10Go). Accordingly, current models suggest a recessive nature for the genomic instability present in HNPCC tumorigenesis associated with the lack of microsatellite alterations in normal tissues from HNPCC patients (6Go,13Go,14Go). We have used an alternative PCR-cloning approach to further investigate, at the level of single microsatellite molecules, whether microsatellite instability can occur before tumor diagnosis and thus be helpful in identifying HNPCC carriers.

Because of the mutational dynamics of repetitive sequences, genomic microsatellites show a polymorphic length distribution that depends on their inherent instability and the status of the MMR system. Even when the MMR system is functional, a small percentage of mutations escape from repair and become clonally perpetuated, allowing microsatellites to exhibit different lengths in a gaussian-like distribution even in normal tissues (22Go,23Go). Our aim was to characterize the distribution patterns of bat26 single molecules in peripheral blood lymphocytes (PBLs) from non-carrier and carrier members of two HNPCC families with known germline mutations in MLH1 (Family A, with a G>A missense mutation in codon 67 of exon 2) (Fig. 1) and in MSH2 (Family B, with a G insertion in nucleotide 735 of exon 4). A total of 901 single molecules from four non-carrier members (three members of Family A and 266 molecules from one member of Family B) were analyzed. As expected, distributions showed a gaussian-like pattern of bat26 with lengths ranging from 21 to 27 adenines that we considered as stable alleles (Table 1). Interestingly, the most common length of bat26 was 25 adenines instead of 26 in all cases. No differences were detected with respect to age. These patterns were considered as normal for comparison purposes.



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  		Figure 1. Family A is represented. This family shows a positive family history for colorectal cancer and is also positive for Amsterdam criteria (30Go). Accordingly, it was diagnosed as an HNPCC family and few carriers with a germline mutation in the MLH1 gene were identified. Carrier and non-carrier members are indicated, as well as members with colorectal cancer. The members selected for the bat26 analysis are numbered accordingly. Cases 317, 649 and 825 were healthy at the time of blood extraction.

 

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  		Table 1. Bat26 allelic profiles in PBLs from carriers and non-carriers from HNPCC families
 
Next, we studied the allelic distribution of bat26 alleles present in PBLs of five phenotypically normal (at the time of blood extraction) HNPCC carriers from Family A and one carrier from Family B (Table 1 and Fig. 2 A and C). All carriers showed a gaussian-like pattern with specific altered microsatellite molecules (20 adenines tracts or shorter) (number of 20A–14A alleles was 0 of 1167 in controls versus 81 of 1566 in carriers, P<0.001). The overall frequency of altered bat26 sequences (unstable alleles) in carriers was 5.6% (102/1814) (range: 3.53–7.09) of the total number of cloned molecules (Table 1). Again, no differences were detected with respect to regarding age. Further experiments with a single bat26 clone demonstrated that altered lengths were not owing to artifacts from our PCR-cloning approach (Fig. 2b). These results suggest that subjacent low-level genomic instability characterizes HNPCC carriers and that this instability may occur well before tumor diagnosis.



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  		Figure 2. Bat26 mutations in HNPCC carriers. (A) An illustrative figure of bat26 clones obtained from the HNPCC-carrier 768 is shown. The respective sizes of the adenine repeats are depicted on top. (B) A representative autoradiography showing the absence of mutational events caused by the PCR-cloning approach when using a single clone of 20 adenines as control. (C) The sequence data from one of the (A)18 clones found in the HNPCC-carrier 768 are shown.

 
The molecular basis of the observed low-level genetic instability detected in phenotypically normal HNPCC carriers may have distinct explanations. One possibility is that they reflect the presence of circulating phenotypically normal MSI(–/+) cells. Several theoretical explanations can be envisioned for this phenotype. On the one hand, a reduced dose (haploinsufficiency) of effective MMR molecules could explain it. This would be in agreement with previous observations suggesting that, in non-neoplastic HNPCC colon cells, oncogenic mutations might accumulate in tissues before a complete loss of MMR occurs (19Go). This suggestion is in line with previous results with distinct animal models. Heterozygous mutants of MSH2 in Drosophila germ cells also show MSI in the germline (24Go,25Go). Furthermore, colon tissues from heterozygous MSH2 transgenic mice are characterized by increased levels of genomic frameshift deletions (26Go) and similar data have been obtained from defective MSH2 and MLH1 yeast strains (27Go,28Go). In this case, the somatic mutations observed would accumulate from birth in tissues of HNPCC carriers. While a role for insufficiency is provocative, it certainly does not account for all our findings. In fact, age of carrier does not correlate with the degree of instability, as would be expected under haploinsufficiency. Another possibility is that dominant-negative effect of both MLH1 and MSH2 mutations exists in normal MSI(+/–) cells. However, this possibility is unlikely. First, because a majority of HNPCC tumors show a second hit affecting the wild-type MMR allele, mainly by loss of heterozygosity, suggesting that most HNPCC MMR mutations do not exert dominant-negative functions. Second, because reported individuals showing homozygous germline mutations in MMR genes died at very young age (17Go).

Another relevant possibility is that the shorter alleles we detect depict the presence of circulating MSI(+/+) cells that have completely lost MMR. The presence of very short alleles (15A–17A) in several of the carriers analyzed, highly suggests the presence of cell populations that have accumulated a high number of alterations. These cells could be either phenotypically normal or already malignant. The fact that four of the five carriers had been diagnosed of neoplasia either before or after blood extractions makes this possibility a feasible one.

These results may have some clinical implications. Detection of low-level MSI may be used in the characterization of members at risk of HNPCC families in families with MSI(+) tumors with no detectable germline mutations in the known MMR genes. Also, the detection of rare mutant bat26 alleles might be an additional tool for evaluating the pathogenic capabilities of missense MMR mutations in HNPCC carriers, although it does not alone prove pathogenicity. The missense mutation from G to A at codon 67 (GGG to AGGG; G67R) of Family A is worthy of note. This mutation has been considered as pathogenic based on three facts: (a) this glycine residue is highly conserved, (b) the mutation has not been found in control individuals and the mutation (c) cosegregates with the disease. The experimental evidence we report here should be used as further evidence supporting its pathogenicity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Sample collection and DNA extractions
Genomic DNA samples from PBLs were obtained from previously characterized members of two HNPCC families with known germline MMR mutations attended at the Cancer Genetic Unit at the Institut Català d'Oncologia, (Barcelona, Spain). Five HNPCC carriers and three non-carriers of Family A (Fig. 1) harboring a G199A missense mutation in codon 67 of exon 2 of MLH1 and two members—one carrier and one non-carrier—of Family B, with a frameshift mutation in MSH2 produced by a G insertion at nucleotide 735 of exon 4, were included. The G199A mutation has been previously described and is considered as a pathogenic mutation (see the International Society of Gastrointestinal Hereditary Tumours Mutation database, http://www.insight-group.org).

All included patients had undergone endoscopic procedures 1–2 years before blood extraction. At the time of blood extraction, all colonoscopies were negative. Of all included patients, one carrier (825+) had been diagnosed of a colorectal carcinoma 1 year before. He had no evidence of disease at the time of blood analysis. Another carrier (376+) had been diagnosed of an ovarian cancer 2 years before blood extraction and of a colorectal cancer 1 year before. Follow-up period oscillated between 4 and 8 years. During follow-up, and in the absence of disease, colonoscopic procedures have been performed every 2 years before 40 years of age and yearly afterwards. Current follow-up status is depicted in Table 1. Case 317 was diagnosed of adenoma 2 years after blood extraction and a carcinoma was diagnosed 3 years later.

DNA was extracted as described. Briefly, blood samples were treated with erythrocyte lysis buffer (2 M Tris, p. 7.5; 1 M MgCl2), during 30 min on ice and supernatants recovered by centrifugation. Leukocyte lysis buffer (5 M NaCl; 0.25 M EDTA, pH. 8.0; 2 M Tris, p. 7.5) and proteinase K were added to each tube and samples incubated during 5 h at 50 and at 37°C overnight. DNA was extracted with chloroform and isopropanol using standard procedures. Sample collection was carried out in accordance with previously established ethical protocols.

Analysis of bat26 allelic profiles
The microsatellite bat26 was amplified by PCR as previously described (20), and PCR products cloned into PCR4-Topo vector (Invitrogen, Carlsbad, CA, USA). Transformed Escherichia coli were grown with ampicillin at 37°C and colonies picked after 12 h. Clones were reamplified by radioactive PCR in the presence of 0.2 mCi of [{alpha}-32P]dCTP as follows: one cycle at 94°C for 4 min., followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR products were analyzed on a 6% denaturing polyacrylamide gel and subjected to autoradiography (29Go). A previously characterized clone of bat26 of 17 adenines was used as control. The size of every clone was determined by band counting, using the size of the control as reference. In order to ensure that the assigned sizes were as expected, a representative percentage of randomly selected clones were also sequenced on an ABI Prism 377 automatic sequencer (Perkin-Elmer, Foster City, CA, USA) using the ABI Prism Dye Terminator Cycle Sequencing Kit (Perkin-Elmer). All sequences confirmed the expected sizes. Proportions of alleles were compared by means of Yates' corrected Pearson chi-square test. Furthermore, we used a bat26 clone of 20 adenines to address whether the PCR-cloning technique of our approach could yield artifactual shorter alleles of bat26 that will invalidate the analysis. This clone was therefore reamplified, cloned again into PCR4-Topo vector and grown in bacteria. Fifty colonies were randomly selected and analyzed accordingly. We did not detect any allelic variant other than the original.


   ACKNOWLEDGEMENTS
 
Supported by grants FIS 01/1350, 01/1264 from the Spanish Fondo de Investigaciones Sanitarias. H.A. and E.D. are supported by fellowships from the Spanish Fondo de Investigaciones Sanitarias.


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 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
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