Microsatellite instability in saliva from patients with hereditary non-polyposis colon cancer and siblings carrying germline mismatch repair gene mutations.

Microsatellites are short tandem repeats of deoxyribonucleic acid (DNA) sequences which are distributed throughout the genome. Tumors in patients with Lynch syndrome tend to accumulate mutations in microsatellites at a much higher rate than other sequences in the genome resulting in microsatellite instability (MSI). This is due to germline mutations in mismatch repair (MMR) genes. Using small pool-polymerase chain reaction (SP-PCR), previous studies have shown that mutant alleles can be detected in microsatellites of DNA from peripheral blood lymphocytes (PBLs) of Lynch syndrome patients at frequencies that were low, but significantly higher than frequencies in PBLs of age-matched non-Lynch syndrome controls. In the present study, SP-PCR detection of frequency of mutant MSI alleles (FMMA) was performed on PBLs and saliva samples from four sets of families. Each family set consisted of a mutation carrying affected proband (initial tumor bearer), a germline mutation-carrier sibling without tumors, and an age-matched normal control, either related (for 3 family sets) without mutation carrier status or unrelated (for 1 family set) without mutation carrier status. FMMAs of saliva and PBL DNA were compared between each proband, sibling and control for each family set, and between family sets. In all five statistically significant saliva comparisons identified between germline mutation carriers (FMMA: 0.080-0.261) and normal controls (FMMA: 0.003-0.087), the measured FMMAs were always higher in the carriers (p < 0.05). A logistic regression model of the data showed a significant increase in FMMAs in saliva DNA from siblings with MMR mutation compared to the normal controls (p < 0.001). These results indicated that the increased FMMAs observed in the saliva DNA as well as PBL DNA of MMR gene mutation carriers compared to normal controls are real and repeatable. Furthermore, the logistic regression also indicated that the FMMAs seen in saliva were nearly double those seen in PBLs (p < 0.001). Saliva testing, a less-invasive procedure than PBL testing, is more sensitive and appears to be a viable alternative for identifying MSI in carriers with MMR mutations.

Microsatellite instability in the peripheral blood leukocytes of HNPCC patients.

Most hereditary nonpolyposis colorectal cancer (HNPCC) patients inherit a defective allele of a mismatch repair (MMR) gene, usually MLH1 or MSH2, resulting in high levels of microsatellite instability (MSI-H) in the tumors. Presence of MSI in the normal tissues of mutation carriers has been controversial. Here we directly compare MSI in the peripheral blood leukocyte (PBL) DNA of seven HNPCC patients carrying different types of pathogenic MMR mutations in MLH1 and MSH2 genes with the PBL DNA of normal age-matched controls and of patients with sporadic colorectal cancer (SCRC). Small pool PCR (SP-PCR) was used studying three microsatellite loci for at least 100 alleles each in most samples. The average frequencies of mutant microsatellite fragments in each HNPCC patient (0.04-0.24) were significantly higher (p<0.01) relative to their age-matched normal controls with mutant frequencies (MF) from 0.00 to 0.06, or SCRC patients (MF from 0.01-0.03). The data support the conclusions that higher MF in the PBL DNA of HNPCC patients is real and reproducible, may vary in extent according to the type of germline MMR mutation and the age of the individual, and provide a possible genetic explanation for anticipation in HNPCC families.

Instability of expanded CAG/CAA repeats in spinocerebellar ataxia type 17.

Trinucleotide repeat expansions are dynamic mutations causing many neurological disorders, and their instability is influenced by multiple factors. Repeat configuration seems particularly important, and pure repeats are thought to be more unstable than interrupted repeats. But direct evidence is still lacking. Here, we presented strong support for this hypothesis from our studies on spinocerebellar ataxia type 17 (SCA17). SCA17 is a typical polyglutamine disease caused by CAG repeat expansion in TBP (TATA binding protein), and is unique in that the pure expanded polyglutamine tract is coded by either a simple configuration with long stretches of pure CAGs or a complex configuration containing CAA interruptions. By small pool PCR (SP-PCR) analysis of blood DNA from SCA17 patients of distinct racial backgrounds, we quantitatively assessed the instability of these two types of expanded alleles coding similar length of polyglutamine expansion. Mutation frequency in patients harboring pure CAG repeats is 2-3 folds of those with CAA interruptions. Interestingly, the pure CAG repeats showed both expansion and deletion while the interrupted repeats exhibited mostly deletion at a significantly lower frequency. These data strongly suggest that repeat configuration is a critical determinant for instability, and CAA interruptions might serve as a limiting element for further expansion of CAG repeats in SCA17 locus, suggesting a molecular basis for lack of anticipation in SCA17 families with interrupted CAG expansion.

Microsatellite instability (MSI) increases with age in normal somatic cells.

Small pool PCR (SP-PCR) is a sensitive method for the detection and quantification of microsatellite instability (MSI) in somatic cells. Here we propose that mutant microsatellite fragments accumulate with age in normal somatic cells and that this increase in MSI can be quantified by SP-PCR. MSI at 6 microsatellite loci was determined by SP-PCR in PBL DNA from 17 "normal" blood bank donors. These individuals varied in age from 20 to 67 y/o. MSI phenotypes were plotted against age in a regression analyses. A positive slope indicated a correlation between age and MSI phenotype (p=0.0006). The mean weighted average mutant frequencies across all loci for all individuals in the age groups (0.009 for 20-30 y/o; 0.019 for 35-50 y/o; 0.034 for 60-70 y/o) were also significantly different from each other (p<0.01). A baseline for increases of MSI with age in human somatic cells was therefore begun and the effectiveness of SP-PCR to evaluate low, but significant, levels of MSI, established.

Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR.

Identifying microsatellite instability (MSI) by partitioning DNA into multiple small pools containing only single genome amounts of DNA results in trapping both progenitor and low-frequency mutant alleles into pools where they can be identified and counted following PCR. Statistical approaches determining both the frequencies and the significant differences between frequencies of these Poisson-distributed alleles are presented. Results indicate a level of sensitivity and quantification not possible by standard PCR methods. Using material from colon cancer patients with high levels of MSI in their tumors, we also present the molecular and robotic methods for carrying out such studies. Validation experiments indicated mutants detectable at frequencies >0.03 above background. Frequencies obtained in tumor tissue (>0.25) met the expectations of the approach. Significant levels of MSI were detected in the constitutive tissue of the patient carrying a germ-line mutation for mismatch repair, suggesting both mechanistic and clinical applications of the procedure.

Age and insertion site dependence of repeat number instability of a human DM1 transgene in individual mouse sperm.

Precise measurement of germline repeat number mutations is important for understanding the molecular etiology of expanded trinucleotide repeat diseases. We used single genome-equivalent PCR of sperm DNA to measure the mutation frequencies in two lines of Dmt transgenic mice containing an expanded CTG.CAG tract on an identical genetic background. Single genome-equivalent PCR indicated that apparent mutational spectra derived in other investigations from PCR of bulk sperm DNA were largely the consequence of PCR stutter and not mutations. Here we show that sperm from 8-week-old Dmt-D mice had a significantly higher mutation frequency (change of >1 repeat) (14.2%) than those of Dmt-E mice of the same age (5.5%), in agreement with pedigree analysis. Furthermore, the mutation frequency in sperm of Dmt-D mice increased significantly with age (28.0% at 17 weeks). The age dependence of the degree of expansion implies that mutations accumulate with time in spermatogenic stem cells. Similar rates of expansion per spermatogenic cycle in man would yield the large expansions observed in human diseases such as myotonic dystrophy type 1. Pedigree data showed a significant age-dependent bias toward repeat contraction in female transmissions and a trend towards expansion with age in male transmissions. Thus, direct single genome-equivalent PCR of the sperm DNA of an individual male appears to predict the distribution of mutant allele sizes that might be inherited by its offspring. In further contrast to a recent report, the sex of the offspring had no detectable effect on the direction of the mutational length change.

Detection of radiation and cyclophosphamide-induced mutations in individual mouse sperm at a human expanded trinucleotide repeat locus transgene.

A method to measure the germline mutations induced by cancer treatment in humans is needed. To establish such a method we used a transgenic mouse model consisting of a human DNA repeat locus that has a high spontaneous mutation frequency as a biomarker. Alterations in repeat number were measured in individual sperm from mice hemizygous for an expanded (CTG)(162) human myotonic dystrophy type 1 (DM1) microsatellite repeat using single genome-equivalent (g.e.) PCR and detection by a DNA fragment analyzer. Mutation frequencies were measured in DNA from sperm from controls and sperm derived from stem spermatogonia, differentiating spermatogonia, and spermatocytes exposed to radiation and from spermatocytes of mice treated with cyclophosphamide. There was no increase above control levels in mutations, scored as >1 repeat changes, in any of the treated groups. However, moderately large deletion mutants (between 9 and 20 repeat changes) were observed at frequencies of 2.2% when spermatocytes were treated with cyclophosphamide and, 1.8 and 2.5% when spermatocytes and stem cells, respectively, were treated with radiation, which were significantly higher than the frequency of 0.3% in controls. Thus, radiation and cyclophosphamide induced deletions in the expanded DM1 trinucleotide repeat. PCR artifacts were characterized in sperm DNA from controls and from mice treated with radiation; all artifacts involved losses of more than 20 DM1 repeats, and surprisingly the artifact frequency was higher in treated sperm than in control sperm. The radiation-induced increase in the frequency of PCR artifacts might reflect alterations in sperm DNA that destabilize the genome not only during PCR amplification but also during early embryonic development.