Francesca Migheli

Genetics, Cytogenetics, and Epigenetics of Colorectal Cancer

Most of the colorectal cancer (CRC) cases are sporadic, only 25% of the patients have a family history of the disease, and major genes causing syndromes predisposing to CRC only account for 5-6% of the total cases. The following subtypes can be recognized: MIN (microsatellite instability), CIN (chromosomal instability), and CIMP (CpG island methylator phenotype). CIN occurs in 80–85% of CRC. Chromosomal instability proceeds through two major mechanisms, missegregation that results in aneuploidy through the gain or loss of whole chromosomes, and unbalanced structural rearrangements that lead to the loss and/or gain of chromosomal regions. The loss of heterozygosity that occur in the first phases of the CRC cancerogenesis (in particular for the genes on 18q) as well as the alteration of methylation pattern of multiple key genes can drive the development of colorectal cancer by facilitating the acquisition of multiple tumor-associated mutations and the instability phenotype.

Association of maternal polymorphisms in folate metabolizing genes with chromosome damage and risk of Down syndrome offspring

We analyzed the role of six common polymorphisms in folate metabolizing genes as possible risk factors for having a child with Down syndrome (DS) in 94 Italian mothers of a DS child (MDS) and 113 matched control mothers, both aged less than 35 years at conception. Investigated polymorphisms include methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, and thymidylate synthase (TYMS) 28bp repeat and 1494del6. We also measured the amount of chromosome damage in peripheral blood lymphocytes of 42 MDS and 41 matched controls, by means of the micronucleus assay, and searched for association between this cytogenetic endpoint and any of the studied polymorphisms. Micronuclei in peripheral blood lymphocytes have been analyzed several years after conception: the mean age at sampling was 45.6+/-11.4 years for MDS and 47.95+/-6.9 years for controls. The combined MTHFR 677TT/MTR 2756AA genotype was associated with increased DS risk (P=0.034), and the combined MTHFR 1298AC/TYMS 2R/2R genotype with reduced risk (P=0.003). Moreover, we observed a significant increased frequency of micronucleated lymphocytes in MDS as compared to controls (P<0.0001) and, in the total population, a significant correlation between micronucleated cells and both MTHFR 677C>T (P=0.031) and 1298A>C (P=0.047) polymorphisms.

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing.

Folate, homocysteine, vitamin B12, and polymorphisms of genes participating in one-carbon metabolism in late-onset Alzheimer's disease patients and healthy controls.

AIMS We screened 378 late-onset Alzheimers disease (LOAD) patients and 308 matched controls for the presence of the common MTHFR 677C>T, MTRR 66A>G, MTR 2756 A>G, and TYMS 28 bp repeat polymorphisms, searching for association with disease risk and age at onset. Moreover, we searched for correlation between each of the studied polymorphisms and available data on plasma homocysteine (Hcy), serum folate, and vitamin B12 values. RESULTS We observed a significant increased frequency of the MTHFR 677T allele (0.48 vs. 0.42; p=0.019) and of MTHFR 677CT (OR=1.46; 95%CI=1.03-2.06) and TT genotypes (OR=1.62; 95%CI=1.05-2.49) in LOAD subjects with respect to controls. We also observed a significant increased frequency of the MTRR 66G allele (0.49 vs. 0.43; p=0.044) and of the MTRR 66GG genotype (OR=1.57; 95%CI=1.01-2.46) in the LOAD group. Significantly increased mean plasma Hcy levels (22.7±1.7 vs 14.5±1.7 μmol/L; p=0.037) and decreased serum folate values (5.7±0.5 vs. 7.8±0.8 ng/mL; p=0.005) were observed in LOAD subjects with respect to controls, whilst the difference in serum vitamin B12 values did not reach statistical significance. Several interactions between the studied polymorphisms and biochemical biomarkers were observed. None of the studied polymorphisms was associated with disease age at onset. INNOVATION The present study suggests that the MTRR 66G allele might contribute to LOAD risk and confirms an increased frequency of the MTHFR 677T allele in LOAD. CONCLUSION Overall, present results support a contribution for one-carbon metabolism to LOAD pathogenesis.

Polymorphisms in folate-metabolizing genes, chromosome damage, and risk of Down syndrome in Italian women: identification of key factors using artificial neural networks

BackgroundStudies in mothers of Down syndrome individuals (MDS) point to a role for polymorphisms in folate metabolic genes in increasing chromosome damage and maternal risk for a Down syndrome (DS) pregnancy, suggesting complex gene-gene interactions. This study aimed to analyze a dataset of genetic and cytogenetic data in an Italian group of MDS and mothers of healthy children (control mothers) to assess the predictive capacity of artificial neural networks assembled in TWIST system in distinguish consistently these two different conditions and to identify the variables expressing the maximal amount of relevant information to the condition of being mother of a DS child.The dataset consisted of the following variables: the frequency of chromosome damage in peripheral lymphocytes (BNMN frequency) and the genotype for 7 common polymorphisms in folate metabolic genes (MTHFR 677C>T and 1298A>C, MTRR 66A>G, MTR 2756A>G, RFC1 80G>A and TYMS 28bp repeats and 1494 6bp deletion). Data were analysed using TWIST system in combination with supervised artificial neural networks, and a semantic connectivity map.ResultsTWIST system selected 6 variables (BNMN frequency, MTHFR 677TT, RFC1 80AA, TYMS 1494 6bp +/+, TYMS 28bp 3R/3R and MTR 2756AA genotypes) that were subsequently used to discriminate between MDS and control mothers with 90% accuracy. The semantic connectivity map provided important information on the complex biological connections between the studied variables and the two conditions (being MDS or control mother).ConclusionsOverall, the study suggests a link between polymorphisms in folate metabolic genes and DS risk in Italian women.

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics, and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.

The hOGG1 Ser326Cys polymorphism and Huntington's disease

Increasing evidence supports a role for oxidative DNA damage and impaired DNA repair mechanisms in the pathogenesis of age related neurodegenerative diseases. Within this context there is a current interest in the understanding of the role played by polymorphisms of DNA repair genes in the inter-individual risk to develop neurodegenerative pathologies, as well as in the onset and the progression of disease symptoms. Particularly, somatic CAG repeat expansion of the gene encoding for huntingtin has been observed in tissues of patients affected by Huntingtons disease (HD), including blood and brain. Recent evidence suggests that somatic CAG repeat expansion in HD cells might contribute to disease age at onset and is mediated by the DNA repair OGG1 enzyme, during the removal of 8-oxoguanine (8-oxoG) from the DNA. There is also evidence that the expression of hMTH1, which removes 8-oxoG from the nucleotide pool, protects mice from HD-like symptoms, and progenitor striatal cells from the toxic effects of the mutant huntingtin. The hOGG1 Ser326Cys polymorphism results in reduced OGG1 activity and increased 8-oxoG formation. In the present study, performed on blood DNA from 91 HD subjects, we observed that bearers of the mutant Cys326 allele (Ser326Cys+Cys326Cys) tend to have an increased number of CAG repeats of the expanded HD allele (P=0.049); moreover bearers of at least one copy of the mutant Cys326 allele, mainly heterozygous subjects, showed a significant (P=0.041) earlier disease onset than Ser326Ser wild-type individuals, suggesting a possible role of the hOGG1 Ser326Cys polymorphism in HD phenotype.

DNMT3B Promoter Polymorphisms and Risk of Late Onset Alzheimer’s Disease

The vast majority of Alzheimers disease (AD) are late-onset forms (LOAD) likely due to the contribution of genetic, environmental, and stochastic factors, superimposed on a physiologically age-related decline of neuronal functions. Increasing evidence indicates epigenetic modifications in LOAD brains, and many of the environmental factors associated with AD risk, such as heavy metals and dietary factors, are able to modify the epigenome. There is also indication that environmentally-induced early life modifications of the genome during embryogenesis and brain development could contribute to the development of the disease later in life. DNA methyltransferase 3b (DNMT3b) is an enzyme involved in de novo methylation of the genome during embryogenesis, expressed in progenitor cells during neurogenesis. In the present study we evaluated two functional DNMT3B promoter polymorphisms, namely -149 C > T (rs2424913) and - 579 G > T (rs1569686), as candidate LOAD risk factors. Our analysis of 376 Italian LOAD patients and 308 matched controls revealed no difference in allele frequencies between the case an the control group (OR = 1.10 (0.88-1.39) for rs2424913, and OR = 1.02 (0.81-1.28) for rs1569686). Also the genotype distributions of both polymorphisms were closely similar between groups, and no significant effect on disease age at onset was observed. Overall, present results do not support a major role for rs2424913 or rs1569686 in LOAD pathogenesis.

Susceptibility to Aneuploidy in Young Mothers of Down Syndrome Children

We recently observed an increased frequency of binucleated micronucleated lymphocytes in women who had a Down syndrome (DS) child before 35 years of age and the fluorescence in situ hybridization analysis revealed that micronuclei were mainly originating from chromosomal malsegregation events, including chromosome 21 malsegregation. That study indicated that women who have a DS child at a young age might have a genetic predisposition to chromosome malsegregation in both somatic and germ line cells. Further studies from our group confirmed increased chromosome damage in blood cells of women who had a DS child at a young age and pointed to a possible role for polymorphisms in folate-metabolizing genes in affecting both chromosome damage and DS risk. In the present article, we review the most recent findings on mechanisms and risk factors for chromosome 21 nondisjunction that lead to DS. Multiple risk factors are likely involved in chromosome nondisjunction; they act at different times in the meiotic process and can be of genetic or environmental (epigenetic) origin. We also discuss the increased risk of developing Alzheimers disease (AD) later in life that was observed in women who had a DS child at a young age. Studies performed in the last years that have shown that the brain is, in fact, a complex genetic mosaic of aneuploid and euploid cells support the unified hypothesis trying to relate DS, trisomy 21, and AD.

The hOGG1 Ser326Cys polymorphism is not associated with sporadic Parkinson's disease

Parkinsons disease (PD) is one of the most common neurodegenerative disorders characterized by progressive and profound loss of dopaminergic neurons in the substantia nigra (SN) resulting in resting tremor, rigidity, bradykinesia, and postural instability. The primary cause of the disease is still unknown, but mitochondrial dysfunction and oxidative stress have been implicated in the neurodegenerative process. Oxoguanine DNA glycosylase (OGG1) removes oxidized guanine (8-oxo-G) from the DNA, thus reducing the mutagenic potential of this modified base. Increased 8-oxo-G levels and up-regulation of OGG1 have been detected in the SN of PD brains. Moreover, studies performed in OGG1 knockout mice revealed the importance of this enzyme in protecting dopaminergic neurons against the accumulation of oxidative DNA damage. A common Ser326Cys polymorphism is known in the human gene encoding OGG1 (hOGG1), and the mutant Cys326 variant has been associated with reduced glycosylase activity. In the present study we screened 139 sporadic PD patients and 211 healthy matched controls for the presence of the hOGG1 Ser326Cys polymorphism. The Cys326 allele frequency was similar between the groups (0.20 in PD patients and 0.19 in controls; p=0.817), and no difference in genotype frequencies was observed. Moreover, the hOGG1 Ser326Cys polymorphism was not associated with disease age at onset (p=0.791). Overall, present results suggest that the hOGG1 Ser326Cys polymorphism is not associated with sporadic PD.