Meta- and pooled analyses of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and colorectal cancer: a HuGE-GSEC review.

Worldwide, over 1 million cases of colorectal cancer (CRC) were reported in 2002, with a 50% mortality rate, making CRC the second most common cancer in adults. Certain racial/ethnic populations continue to experience a disproportionate burden of CRC. A common polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene has been associated with a lower risk of CRC. The authors performed both a meta-analysis (29 studies; 11,936 cases, 18,714 controls) and a pooled analysis (14 studies; 5,068 cases, 7,876 controls) of the C677T MTHFR polymorphism and CRC, with stratification by racial/ethnic population and behavioral risk factors. There were few studies on different racial/ethnic populations. The overall meta-analysis odds ratio for CRC for persons with the TT genotype was 0.83 (95% confidence interval (CI): 0.77, 0.90). An inverse association was observed in whites (odds ratio = 0.83, 95% CI: 0.74, 0.94) and Asians (odds ratio = 0.80, 95% CI: 0.67, 0.96) but not in Latinos or blacks. Similar results were observed for Asians, Latinos, and blacks in the pooled analysis. The inverse association between the MTHFR 677TT polymorphism and CRC was not significantly modified by smoking status or body mass index; however, it was present in regular alcohol users only. The MTHFR 677TT polymorphism seems to be associated with a reduced risk of CRC, but this may not hold true for all populations.

The p53 codon 72 variation is associated with the age of onset of hereditary non-polyposis colorectal cancer (HNPCC).

The polymorphic variants at codon 72 of the p53 gene were shown to be functionally distinct in vitro, whereby the arginine (arg) variant induces apoptosis more efficiently than the proline (pro) variant. From the evidence that the DNA mismatch repair system and p53 interact to maintain genomic integrity, we hypothesized that the codon 72 variation may influence the age of onset of disease in HNPCC patients. We tested 538 patients for p53 codon 72 variants, including 167 unrelated patients with pathogenic germline mutations in MSH2 or MLH1 and colorectal carcinoma as first tumour, 126 patients with sporadic microsatellite stable colorectal cancers, and 245 healthy controls. The median age of onset was 41, 36, and 32 years for MSH2 or MLH1 mutation carriers with arg/arg, arg/pro, and pro/pro genotypes, respectively. The log rank test revealed significant differences in the age of onset between arg/arg and pro/pro individuals (p = 0.0002) and in arg/pro versus arg/arg and pro/pro individuals (p = 0.0026 and p = 0.0217, respectively). A Cox regression model indicated an additive mode of inheritance. No significant differences in age of onset were observed among different genotype carriers with microsatellite stable tumours. Our results suggest that p53 codon 72 genotypes are associated with the age of onset of colorectal carcinoma in a mismatch repair deficient background in a dose dependent manner. These findings may be relevant for preventive strategies in HNPCC.

The putative tumor suppressor gene FHIT at 3p14.2 is rarely affected by loss of heterozygosity in primary human brain tumors.

To elucidate the role of the recently identified FHIT gene, located at 3p14.2 in human brain tumor carcinogenesis, a total of 259 tumors were analyzed for loss of heterozygosity (LOH) at microsatellite loci D3S1313, D3S1234, D3S1300, and D3S1481. In primary brain tumors, LOH was detected at a frequency of 8.4% (n = 214). Low-grade gliomas exhibited insignificantly lower LOH rates in comparison to high-grade gliomas (5.3%, n = 19, versus 11.1%, n = 90). Notably, no allelic loss was observed in 12 recurrent glioblastomas analyzed in comparison to their corresponding primary tumor lesions and in two astrocytomas with progression to higher grades of malignancy. Our data indicate that allelic loss of the FHIT gene is neither a critical event in carcinogenesis of primary brain tumors nor tumor grade-associated in astrocytic tumors. In contrast, observed LOH rate for brain metastases was as high as 54.5% (n = 45), in accordance with data thus far accumulated from analyses of corresponding primary tumors.

A microsatellite map of wheat.

Hexaploid bread wheat (Triticum aestivum L. em. Thell) is one of the world's most important crop plants and displays a very low level of intraspecific polymorphism. We report the development of highly polymorphic microsatellite markers using procedures optimized for the large wheat genome. The isolation of microsatellite-containing clones from hypomethylated regions of the wheat genome increased the proportion of useful markers almost twofold. The majority (80%) of primer sets developed are genome-specific and detect only a single locus in one of the three genomes of bread wheat (A, B, or D). Only 20% of the markers detect more than one locus. A total of 279 loci amplified by 230 primer sets were placed onto a genetic framework map composed of RFLPs previously mapped in the reference population of the International Triticeae Mapping Initiative (ITMI) Opata 85 x W7984. Sixty-five microsatellites were mapped at a LOD >2.5, and 214 microsatellites were assigned to the most likely intervals. Ninety-three loci were mapped to the A genome, 115 to the B genome, and 71 to the D genome. The markers are randomly distributed along the linkage map, with clustering in several centromeric regions.

Low-level microsatellite instability phenotype in sporadic glioblastoma multiforme.

PURPOSE: Genetic instability is a hallmark of glioblastoma multiforme (GBM). Microsatellite instability (MSI) is a significant event in the tumorigenesis of many sporadic malignancies. The aim of our investigation was to study microsatellite instability in newly diagnosed glioblastomas. METHODS: MSI was investigated in 109 GBMs with 15 microsatellite markers. Immunohistochemistry was performed for the mismatch repair (MMR) proteins hMLH1, hMSH2, hPMS2, and hMSH6 in cases showing MSI. Sequence and promoter methylation status of hMLH1 were analyzed in the case of a decreased hMLH1 protein expression as well. To further investigate MSI(+) GBMs we carried out studies of LOH at selected chromosome regions, EGFR amplification, and sequence of p53 and PTEN. RESULTS: MSI was observed in six GBMs (5.5%) and it was more frequent in GBMs with a previous lower grade astrocytoma (18.8% vs. 3.2%). MMR protein staining was positive in all MSI(+) GBMs except in one case, which showed an aberrant expression of hMLH1 and hPMS2 without hMLH1 inactivation. Among MSI(+) GBMs, one tumor corresponded to the GBM molecular type 1 (p53 mutation, no EGFR amplification), another tumor to type 2 (wild-type p53, EGFR amplification), and four tumors to neither type (wild-type p53, no EGFR amplification). None of the six tumors carried a PTEN mutation. CONCLUSIONS: MSI in GBM might be caused by inactivation of minor MMR genes rather than by a deficiency of hMLH1 or hMSH2 and it appears not to play a decisive role in the pathogenesis of these tumors. MSI(+) GBMs predominantly showed a profile which included wild-type of p53 and PTEN and absence of EGFR amplification but MSI occurred in all GBM molecular subtypes.

Genomic rearrangements of hMSH6 contribute to the genetic predisposition in suspected hereditary non-polyposis colorectal cancer syndrome.

BACKGROUND: Germline mutations in mismatch repair genes, mainly in hMLH1, hMSH2, and hMSH6, predispose to the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. A substantial fraction of these mutations exists in genomic rearrangements of hMSH2 and hMLH1. In contrast, genomic rearrangements have not been reported in hMSH6. METHODS: Out of 15 HNPCC or HNPCC-like patients who developed tumours with loss of hMSH6 protein expression, we selected three patients who still had no germline mutations after gene sequencing. Genomic DNA of these patients was analysed using PCR based relative quantification of hMSH6 fragments. Indicated exon deletions and amplifications were characterised by long range PCR and sequencing. RESULTS: Genomic rearrangements were identified in two of the three patients. Breakpoint analyses showed an Alu repeat mediated deletion of 13.0 kb affecting the promoter region, exon 1, and exon 2 in one patient, and a duplication of 4.9 kb containing 1.6 kb of the 3' end of exon 4 and exon 5, integrated into intron 5, in the other patient. CONCLUSIONS: Although genomic rearrangements of hMSH6 only play a small role in the spectrum of all mutations predisposing to HNPCC, our results suggest that up to 10-20% of patients with hMSH6 negative tumours harbour germline rearrangements in this gene.

An intronic germline transition in the HNPCC gene hMSH2 is associated with sporadic colorectal cancer.

The aim of this study was to determine whether an intronic germline substitution in the hereditary non-polyposis colorectal cancer (HNPCC) gene hMSH2 represents a genetic risk factor for sporadic CRC. Possible effects of this substitution were investigated by assessment of microsatellite instability and hMSH2 cDNA sequencing. Constitutional DNA from patients with sporadic CRC and healthy controls from the same region in Germany was analysed for the intronic germline T-->C transition six bases upstream of exon 13 of hMSH2. 29 of 106 patients (27%) were found to harbour the germline T-->C transition as opposed to only 13 of 125 controls (10%; P < 0.001; OR 3.2, CI 1.58-6.63). CRCs from patients with the substitution displayed neither clinical HNPCC-like features nor an increased rate of microsatellite instability. No abnormal cDNA sequence was found at the exon 12-13 border. These data suggest a 3.2-fold increased risk of sporadic CRC for individuals with the intronic hMSH2 transition. However, this substitution might not be pathogenic itself, but may be linked to a locus nearby that is.

Doublex sequencing in molecular diagnosis of hereditary diseases.

We describe doublex sequencing of human genomic PCR products using two differently labeled primers in a single reaction and analysis on two automated DNA sequencing devices. Feasibility of the methodology is demonstrated by isothermal and cycle sequencing for two different PCR products and by cycle sequencing on both strands of a single product. It was applied to analyze mutations in patient DNAs in routine sample screening. Because it has the advantage of increased throughput and cost reduction while retaining its accuracy and reading length, we found that doublex sequencing is an attractive option for molecular diagnosis of hereditary diseases. This approach would be even more beneficial if it used DNA sequencing devices with several lasers in a single instrument.

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2.

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.

Detection of genetic diversity in closely related bread wheat using microsatellite markers.

Wheat microsatellites (WMS) were used to estimate the extent of genetic diversity among 40 wheat cultivars and lines, including mainly European elite material. The 23 WMS used were located on 15 different chromosomes, and revealed a total of 142 alleles. The number of alleles ranged from 3 to 16, with an average of 6.2 alleles per WMS. The average dinucleotide repeat number ranged from 13 to 41. The correlation coefficient between the number of alleles and the average number of repeats was only slight (r s = 0.55). Based on percentage difference a dendrogram is presented, calculated by the WMS-derived data. All but two of the wheat cultivars and lines could be distinguished. Some of the resulting groups are strongly related to the pedigrees of the appropriate cultivars. Values for co-ancestry (f) of 179 pairs of cultivars related by their pedigrees (f[Symbol: see text]0.1) averaged 0.29. Genetic similarity (GS) based on WMS of the same pairs averaged 0.44. The rank correlation for these pairs was slight, with r s = 0.55, but highly significant (P<0.001). The results suggest that a relatively small number of microsatellites can be used for the estimation of genetic diversity and cultivar identification in elite material of hexaploid bread wheat.

Quantitative differences between aberrant transcripts which occur as common isoforms and due to mutation-based exon skipping of the mismatch repair gene hMLH1.

About one-third of hereditary non-polyposis colorectal cancer-related mutations in the mismatch repair gene hMLH1 result in the loss of entire exons from the wild type transcripts. Here we describe quantitative differences of hMLH1 transcripts without exon 15, exon 16 or exon 17 in several members of a family with hereditary non-polyposis colorectal cancer. The transcript lacking exon 15 is caused by a G to A transition affecting the last nucleotide of the respective exon and results in a truncated protein. The transcripts lacking exon 16 or exon 17, which are in-frame deletions, were also found in all tested samples of a normal population and represent common isoforms. Reverse transcription-polymerase chain reaction-based relative quantification revealed about 50 % signal intensity for the mutation-based transcript, but less than 10% for the common isoforms, if compared to the wild type. All aberrant transcripts were detected from blood-derived cDNAs but not from samples of normal colon epithelium. Although the biological significance of the common isoforms is unknown, they might lead to false risk assessment in hereditary non-polyposis colorectal cancer cases.

[Sequence analysis of BRCA1 gene in young breast cancer patients and/or positive family history]. / Sequenzanalyse des BRCA1 Gens bei Brustkrebspatientinnen mit jungem Erkrankungsalter und/oder positiver Familienanamnese.

We analyzed the complete coding region with adjacent intron sequences of the BRCA1 gene in eight patients with hereditary and/or early-onset breast/ovarian cancer. We detected one germline mutation in exon 5 in a 35-year-old woman with early-onset breast and ovarian cancer and 10 polymorphisms, of which one has not been published so far. To determine whether certain BRCA1 polymorphisms are associated with an increased risk for breast cancer, we will compare genotype distributions of early-onset breast cancer populations with matched controls.

RFLP mapping of genes affecting plant height and growth habit in rye.

RFLP mapping of chromosome 5R in the F3 generation of a rye (Secale cereale L.) cross segregating for gibberellic acid (GA3)-insensitive dwarfness (Ct2/ct2) and spring growth habit (Sp1/sp1) identified RFLP loci close to each of these agronomically important genes. The level of RFLP in the segregating population was high, and thus allowed more than half of the RFLP loci to be mapped, despite partial homozygosity in the parental F2 plant. Eight further loci were mapped in an unrelated F2 rye population, and a further two were placed by inference from equivalent genetic maps of related wheat chromosomes, allowing a consensus map of rye chromosome 5R, consisting of 29 points and spanning 129 cM, to be constructed. The location of the ct2 dwarfing gene was shown to be separated from the segment of the primitive 4RL translocated to 5RL, and thus the gene is probably genetically unrelated to the major GA-insensitive Rht genes of wheat located on chromosome arms 4BS and 4DS. The map position of Sp1 is consistent both with those of wheat Vrn1 and Vrn3, present on chromosome arms 5AL and 5DL, respectively, and with barley Sh2 which is distally located on chromosome arm 7L (= 5HL).

Evidence that TSG101 aberrant transcripts are PCR artifacts.

Critical analysis of the data published so far concerning the TSG101 gene revealed some inconsistencies leading us to its re-evaluation in 80 breast, brain, colon, and neuroectodermal tumors and 37 normal tissue specimens. In this study, the occurrence of TSG101 cDNA aberrant transcripts was verified, but in addition we made observations that are in apparent conflict with the aberrant splicing theory supposed as the underlying mechanism for transcript formation: the location of most deletion breakpoints within exons and nonconformity of these putative splice sites with the highly conserved GT-AG rule, detection of insertions as well as nonreproducible and highly variable results in repeated RT-nested PCRs. Furthermore, we found that reamplification of full-length TSG101 cDNA products leads to the generation of deleted transcripts. In summary, for the first time we provide evidence that the acquired TSG101 transcripts are caused by PCR artifacts.

[Molecular diagnosis and clinical consequences in families with HNPCC syndrome]. / Molekulare Diagnostik und klinische Konsequenzen bei Familien mit HNPCC-Syndrom.

Thirteen families were included in our HNPCC surveillance program, eight of which met strict and three incomplete Amsterdam criteria. Two index patients were younger than 35 years of age. Tumors of all index persons showed microsatellite instabilities in at least 50% of the ten markers studied. Immunohistochemical analysis of tumor sections was performed using antibodies against hMLH1 and hMSH2 proteins in order to identify the mutated gene, which is not expressed in the tumor. Coding regions of hMLH1 and hMSH2 genes were amplified by PCR from genomic DNA and sequenced. In nine families pathogenetic mutations all resulting in a truncated protein, could be identified. Furthermore, 21 intron and exon polymorphisms were found. Since July 1997 we have offered surgical and genetic counseling to families with hereditary cancer syndromes in a special outpatient clinic. In addition to 13 index patients three asymptomatic gene carriers were included and eight noncarriers were excluded from the HNPCC surveillance program, as recommended by the HNPCC study group of Germany. Molecular diagnosis has considerable clinical implication in hereditary nonpolyposis colorectal cancer (HNPCC) families with respect to family members who actually have the disease as well as gene carriers and noncarriers.

Clinical consequences of molecular diagnosis in families with mismatch repair gene germline mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC), clinically defined by the Amsterdam criteria, is associated with mismatch repair gene germline mutations. This study was performed to evaluate the efficiency of combined clinical and molecular diagnostics in identifying carriers of a mutated gene in families meeting criteria of the Bethesda guidelines and to examine the influence of molecular diagnosis on clinical decision-making in carriers and noncarriers. Seventy-two patients meeting criteria of the Bethesda guidelines were tested for microsatellite instabilities (MSI). MSI-H tumors were found in 38 (52.8%) index patients. Complete sequencing of hMLH1 and hMSH2 in 38 MSI-H patients and of hMSH6 in one of these patients revealed 15 pathogenic germline mutations, including three novel mutations, and three novel unclassified germline variants. Twelve of the 15 pathogenic mutations were found in patients fulfilling the Amsterdam I/II criteria. Surgical and genetic counseling was offered to the affected families; as a result of molecular diagnosis in the 15 families, 26 index patients and affected carriers and 8 asymptomatic carriers of a mutated mismatch repair gene were included in the surveillance program, and 26 noncarriers were excluded from this program. Although germline mutations are detected in only 20.8% of patients fulfilling criteria of the Bethesda guidelines, family history and MSI-H tumor classification are both strong indicators for germline mutations in hMSH2, hMLH1, and hMSH6 genes, resulting in a 51.9% mutation detection rate. Identification of individual mutation status allows clear-cut decisions on whether or not inclusion in surveillance programs is indicated.

Combined molecular and clinical approach for decision making for surgery in HNPCC patients: a report on three cases in two families.

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with highly penetrant germline mutations in mismatch repair genes. Due to a high lifetime risk in gene carriers for synchronous and for metachronous colorectal cancer and endometrial cancer in women, prophylactic and extended surgery are considered as options for gene carriers. A 54-year-old patient with a history of metachronous rectal cancer and a family history fulfilling the Amsterdam criteria presented with carcinoma of the cecum and highly dysplastic adenomas of the splenic flexure and descending colon. As a result of these findings, medical history and molecular diagnosis, the decision was made to perform colectomy and prophylactic hysterectomy with oophorectomy; histological examination of the specimen showed three synchronous colon carcinomas. The 31-year-old son carrying the pathogenic mutation refused to be included in the HNPCC surveillance program. One year later he presented with symptoms of bowel obstruction, and a carcinoma of the descending colon was diagnosed. Intraoperatively, in addition to the colon cancer, a small bowel cancer and peritoneal carcinomatosis were found. In another family fulfilling the Amsterdam criteria without known germline mutation a woman presented with synchronous cancer of the ascending colon and the lower rectum at the age of 49 years. Proctocolectomy and prophylactic hysterectomy were performed, which revealed an additional colon cancer and endometrial cancer. We discuss approaches for individual decision making for surgery in HNPCC patients. Is a subtotal colectomy indicated in the case of first colon cancer in HNPCC patients, or if the first tumor occurs in the lower rectum, should a proctocolectomy or a restorative proctocolectomy be considered? The aim of prospective clinical studies should be to assess acceptability, survival rates, mortality, and the quality of life in HNPCC patients who have undergone surveillance and standard oncological resections versus extended or prophylactic surgery.

Sequence analysis of the mismatch repair gene hMSH6 in the germline of patients with familial and sporadic colorectal cancer.

To evaluate the involvement of hMSH6 in colorectal cancer, the complete coding sequence and flanking intron regions of the gene were analyzed by DNA sequencing in 10 patients fulfilling Bethesda Guidelines for colorectal tumors and 10 patients with sporadic colorectal carcinoma. In addition, 10 mono- and 10 dinucleotide repeat markers were analyzed for microsatellite instability. A protein-truncating T insertion at codon 218 was identified in the index person of a hereditary non-polyposis colorectal cancer (HNPCC)-like kindred and was accompanied by a somatic T deletion in the tumor. The tumor of this patient was positive for mono- but negative for dinucleotide repeat instability and lacked allelic losses at loci frequently affected in colorectal carcinomas. A novel amino acid change, F340S, was found in a patient with sporadic colon and breast cancer and leukemia but was not detected in 246 chromosomes from healthy anonymous blood donors. In addition, we describe 2 silent and 15 intronic sequence variants not previously reported. Although the frequency is low, we present further evidence for hMSH6 germline mutations that predispose patients to HNPCC-like phenotypes and suggest that mono- and dinucleotide repeat instability testing may be useful for distinguishing between individuals harboring an hMSH2 or hMLH1 mutation and a mutation of the hMSH6 gene.

Abundance, variability and chromosomal location of microsatellites in wheat.

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)n blocks was estimated to be 3.6 x 10(4) and the number of (GT)n blocks to be 2.3 x 10(4) per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.