Founding mutations and Alu-mediated recombination in hereditary colon cancer.

By screening members of Finnish families displaying hereditary nonpolyposis colorectal cancer (HNPCC) for predisposing germline mutations in MSH2 and MLH1, we show that two mutations in MLH1 together account for 63% (19/30) of kindreds meeting international diagnostic criteria. Mutation 1, originally detected as a 165-base pair deletion in MLH1 cDNA comprising exon 16, was shown to consist of a 3.5-kilobase genomic deletion most likely resulting from Alu-mediated recombination. Mutation 2 destroys the splice acceptor site of exon 6. A simple diagnostic test based on polymerase chain reaction was designed for both mutations. Our results show that these two ancestral founding mutations account for a majority of Finnish HNPCC kindreds and represent the first report of Alu-mediated recombination causing a prevalent, dominantly inherited predisposition to cancer.

Primary sequence of ovomucoid messenger RNA as determined from cloned complementary DNA.

Ovomucoid messenger RNA (mRNAom) comprises approximately 8% of the total mRNA in the estrogen-stimulated oviduct. The recombinant plasmid pOM100 contained DNA complementary to the 3' end of mRNAom. DNA complementary to the 5' end of mRNAom was obtained from a partially purified preparation of mRNAom by polymerization by reverse transcriptase in the presence of a restriction fragment primer from pOM100. The complementary DNA mixture was amplified by molecular cloning using poly dG/dC tailing to form recombinant bacterial plasmids. Recombinant plasmids containing ovomucoid DNA sequences were selected by in situ hybridization to 32P-labeled pOM100 fragments. The longest plasmid containing ovomucoid DNA sequences was designated pOM502. The complete DNA sequence of both pOM100 and pOM502 was determined. The two plasmids appear to contain sequences complementary to the entire length of mRNAom. The nucleic acid sequence agrees with the known amino acid sequences for both ovomucoid and its N-terminal signal peptide. Highly homologous sequences occur in two regions that coincide with structural domains of the protein. Comparison of the sequence of mRNAom with that for other eucaryotic mRNAs allowed identification of possible functional regions in the mRNA molecule.

Semiautomated assessment of loss of heterozygosity and replication error in tumors.

Loss of heterozygosity (LOH) and replication error (RER) are important phenomena in tumor development, with diagnostic and prognostic relevance. Therefore, screening for LOH and RER is a desirable first step in the molecular analysis of tumors. We used semiautomated procedures based on multicolor fluorescently labeled microsatellite markers and an automated sequencer for PCR amplification, electrophoresis of PCR products, and allele detection with a set of 16 microsatellites in 56 colorectal tumors. We improved existing software for computer-assisted assessment of LOH and RER. A comparison of these results with those of a conventional, radioactive technique and visual interpretation shows a high degree of correlation between the two methods. The detection rates of LOH and RER are similar to those reported previously. The main advantages of the semiautomated fluorescence-based typing are in the objective, observer-unrelated, easy, and rapid computer-based scoring, and the resulting quantitative assessment of RER.

Microsatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC).

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant cancer predisposition syndrome caused by germ-line mutations in DNA mismatch repair genes. It is relevant to identify HNPCC patients because colonoscopic screening of individuals with HNPCC mutations reduces cancer morbidity and mortality. Microsatellite instability (MSI) is characteristic of HNPCC tumors. A panel of five markers (BAT25, BAT26, D2S123, D5S346, and D17S250, the so-called Bethesda markers) has been proposed for screening for MSI. To test a hypothesis that the use of BAT26 alone is feasible in screening for MLH1/MSH2 mutation-positive HNPCC patients, we compared the MSI results of 494 colorectal cancer patients obtained using BAT26 with results obtained using the Bethesda markers. BAT26 was able to identify all 27 mutation-positive individuals in this series. The marker failed to identify 2 high MSI tumors and 20 low MSI tumors, all of which expressed MLH1, MSH2, and MSH6 when scrutinized by immunohistochemistry.

Mismatch repair genes and mononucleotide tracts as mutation targets in colorectal tumors with different degrees of microsatellite instability.

Microsatellite instability occurs in 15% of colorectal carcinomas and may be due to replication errors (RER). The pattern of instability--'severe' vs 'mild'--and the tumorigenic pathway, as reflected by the involvement of functionally important genes, may vary according to the underlying gene(s). We defined 'mild' RER as mono- or tetranucleotide repeat instability in the absence of widespread instability at dinucleotide repeats and studied 15 colorectal tumors with this phenotype for mutations in the DNA mismatch repair genes MSH2, MLH1, MSH3, and MSH6. No mutations were found, suggesting that these genes were not implicated. We then compared colorectal cancers with 'mild' RER (n = 15), and those with 'severe' RER without (n = 11) or with (n = 22) detectable mutations in MSH2 or MLH1 to assess the involvement of mononucleotide repeats contained in the coding regions of MSH3, MSH6, BAX, and TGFbeta RII. The combined mutation rates of the above mentioned loci varied significantly between the three groups of tumors, being 0%, 25% and 52%, respectively. Furthermore, the individual genes showed specific patterns of involvement; for example, among tumors with 'severe' RER, TGFbeta RII displayed uniformly high mutation rates while MSH3, MSH6, and BAX were more frequently altered in tumors that also showed MSH2 or MLH1 mutations. Our findings suggest that different subcategories exist among unstable tumors, defined by the RER pattern on the one hand and tumorigenic pathway on the other, and structural changes of MSH2 and MLH1 are likely to explain only a proportion of these cases.

Population-based molecular detection of hereditary nonpolyposis colorectal cancer.

PURPOSE: Cancer morbidity and mortality can be dramatically reduced by colonoscopic screening of individuals with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, creating a need to identify HNPCC. We studied how HNPCC identification should be carried out on a large scale in a sensitive and efficient manner. PATIENTS AND METHODS: Colorectal cancer specimens from consecutive newly diagnosed patients were studied for microsatellite instability (MSI). Germline mutations in the MLH1 and MSH2 genes were searched for in MSI(+) individuals. RESULTS: Among 535 colorectal cancer patients, 66 (12%) were MSI(+). Among these, 18 (3.4% of the total) had disease-causing germline mutations in MLH1 or MSH2. Among these 18 patients, five were less than 50 years old, seven had a previous or synchronous colorectal or endometrial cancer, and 15 had at least one first-degree relative with colorectal or endometrial cancer. Notably, 17 (94%) of 18 patients had at least one of these three features, which were present in 22% of all 535 patients. Combining these data with a previous study of 509 patients, mutation-positive HNPCC accounts for 28 (2.7%) of 1,044 cases of colorectal cancer, predicting a greater than one in 740 incidence of mutation-positive individuals in this population. CONCLUSION: Large-scale molecular screening for HNPCC can be done by the described two-stage procedure of MSI determination followed by mutation analysis. Efficiency can be greatly improved by using three high-risk features to select 22% of all patients for MSI analysis, whereby only 6% need to have mutation analysis. Sensitivity is only slightly impaired by this procedure.

Molecular screening for hereditary nonpolyposis colorectal cancer: a prospective, population-based study.

PURPOSE: Germline mutations in mismatch repair genes predispose to hereditary nonpolyposis colorectal cancer (HNPCC). To address effective screening programs, the true incidence of the disease must be known. Previous clinical investigations reported estimates ranging between 0.5% and 13% of all the colorectal cancer (CRC) cases, whereas biomolecular studies in Finland found an incidence of 2% to 2.7% of mutation carriers for the disease. The aim of the present report is to establish the frequency of the disease in a high-incidence area for colon cancer. PATIENTS AND METHODS: Through the data of the local CRC registry, we prospectively collected all cases of CRC from January 1, 1996, through December 31, 1997 (N = 391). Three hundred thirty-six CRC cases (85.9% of the incident cases) were screened for microsatellite instability (MSI) with six to 12 mono- and dinucleotide markers. MSI cases were subjected to MSH2 and MLH1 germline mutation analysis and immunohistochemistry; the methylation of the promoter region was studied for MLH1. RESULTS: Twenty-eight cases (8.3% of the total) showed MSI. MSI cases differed significantly from microsatellite-stable (MSS) cases for their proximal location (P <.01), high mucinous component (P <.01), and poor differentiation (P =.002). Of MSI cases studied (n = 12), only one with a family history compatible with HNPCC had a germline mutation (in MSH2). Five other patients with a family history of HNPCC (two with MSI and three with MSS tumors) did not show germline mutations. CONCLUSION: We conclude that the incidence of molecularly confirmed HNPCC (one [0.3%] of 336) in a high-incidence area for CRC is lower than in previous biomolecular and clinical estimates.

Evidence for two molecular steps in the pathogenesis of myeloid disorders associated with deletion of chromosome 7 long arm.

Partial deletion of the long arm of chromosome 7 (7q-) is a frequent chromosomal aberration in many neoplasias, including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Recurrent deletions, leading to loss of heterozygosity (LOH), may be indicative of a tumor suppressor gene nearby. We studied eight AML or MDS patients with 7q-, in order to define the commonly deleted area in more detail. Separated blood lymphocytes and granulocytes were typed with 48 polymorphic microsatellite markers, and the heterozygosity between the two cell lineages was compared. The minimum commonly deleted region spanned from D7S658 to D7S655. Unexpectedly, four of the patients showed remarkable homozygosity in both lymphocytes and granulocytes around the commonly deleted area, and thus no deletions could be demonstrated by comparing the two cell lineages. Comparison to leukemic patients without 7q- and to normal individuals revealed that the homozygosity was restricted to patients with 7q-. We suggest that a specific mechanism, such as mitotic recombination in bone marrow stem cells, leading to homozygosity in both granulocytes and lymphocytes, represents a leukemogenetic step in these patients.

Genomic structure of the human congenital chloride diarrhea (CLD) gene.

Congenital chloride diarrhea (CLD) is caused by mutations in a gene which encodes an intestinal anion transporter. We report here the complete genomic organization of the human CLD gene which spans approximately 39kb, and comprises 21 exons. All exon/intron boundaries conform to the GT/AG rule. An analysis of the putative promoter region sequence shows a putative TATA box and predicts multiple transcription factor binding sites. The genomic structure was determined using DNA from several sources including multiple large-insert libaries and genomic DNA from Finnish CLD patients and controls. Exon-specific primers developed in this study will facilitate mutation screening studies of patients with the disease. Genomic sequencing of a BAC clone H_RG364P16 revealed the presence of another, highly homologous gene 3' of the CLD gene, with a similar genomic structure, recently identified as the Pendred syndrome gene (PDS).

Consideration of the evolution of the Saccharomyces cerevisiae MEL gene family on the basis of the nucleotide sequences of the genes and their flanking regions.

Analysis of the DNA sequences of new members of the Saccharomyces cerevisiae MEL1-MEL10 gene family showed high homology between the members. The MEL gene family, alpha-galactosidase-coding sequences, have diverged into two groups; one consisting of MEL1 and MEL2 and the other of MEL3-MEL10. In two S. cerevisiae strains containing five or seven MEL genes each, all the genes are nearly identical, suggesting very rapid distribution of the gene to separate chromosomes. The sequence homology and the abrupt change to sequence heterogeneity at the centromere-proximal 3' end of the MEL genes suggest that the distribution of the genes to new chromosomal locations has occurred partly by reciprocal recombination at solo delta sequences. We identified a new open reading frame sufficient to code for a 554 amino acid long protein of unknown function. The new open reading frame (Accession number Z37509) is located in the 3' non-coding region of MEL3-MEL10 genes in opposite orientation to the MEL genes (Accession numbers Z37508, Z37510, Z37511). Northern analysis of total RNA showed no hybridization to a homologous probe, suggesting that the gene is not expressed efficiently if at all.

Characterization of three chicken pseudogenes for U1 RNA.

Three chicken genomic DNA clones containing the U1 RNA sequence were isolated from a chicken gene library and characterized. Two of these clones, CL64 and CL111, are overlapping clones which show several single-nucleotide changes in the U1 coding sequence, suggesting that they probably are alleles of the same sequence. The U1 sequence in the third clone, CL40, is more divergent. Flanking regions of these genes do not share any sequence homology between each other or with the previously isolated chicken genomic clone CL59. A short repeat CGGGG appears 28 times upstream of the U1 sequence in CL59. Another repeat, GCACC, is repeated 14 times upstream of the U1 region in CL40.

A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.

Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley.

The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fairly thermostable, with the optimum temperature being 60 degrees C. It showed maximum activity over a broad pH range (7.0-9.0). The molecular mass of the monomer was about 50,000 Da based on the SDS/PAGE, and the calculated value from the cDNA-deduced polypeptide sequence was 53,620 Da. A relative mass estimation of 100,000 Da was obtained from the Superose 12 chromatography, suggesting that the barley enzyme is a dimer. The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids. The genomic sequence of 4473 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG. One large intron was located in the 5'untranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini. Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.

Ovomucoid intervening sequences specify functional domains and generate protein polymorphism.

Two thirds of the natural chicken ovomucoid gene has been sequenced, including all exons and the intron sequences surrounding all fourteen intron/exon junctions. The junction sequences surrounding four of the introns are redundant; however, the sequences surrounding the other three introns contain no redundancies and thus the splicing sites at either end of these three introns are unambiguous. The splicing in all cases conforms to the GT-AG rule. The ovomucoid gene sequence around intron F can be used to predict the cause of an internal deletion polymorphism in the ovomucoid protein, which is an apparent error in the processing of the ovomucoid pre-mRNA. We also compare the structural organization of the ovomucoid gene with the ovomucoid protein sequence to examine theories of the evolution of ovomucoids as well as the origin of intervening sequences. This analysis suggests that the present ovomucoid gene evolved from a primordial ovomucoid gene by two separate intragenic duplications. Furthermore, sequence analyses suggest that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Finally, the positions of the introns within the ovomucoid gene support the theory that introns separate gene segments that code for functional domains of proteins and provide insight on the manner by which eucaryotic genes were constructed during the process of evolution.

Structure and expression of a chicken gene coding for U1 RNA.

We have isolated and sequenced a genomic fragment containing sequences complementary to chicken U1 RNA. The sequence of this genomic U1 gene is completely homologous and colinear with that of chicken U1 RNA. This U1 gene is part of a multigene family (6--10 copies per haploid genome), and these loci do not appear to be closely clustered. Sequences complementary to other snRNAs are not present within the 2.5 kb genomic fragment containing the U1 gene. We have determined that U1 RNA is synthesized by polymerase II; however, a "Hogness box" is not present upstream from its cap site at the position usually observed for mRNA genes. The synthesis of U1 RNA in oviduct nuclei during different states of hormonal induction also appears to be constitutive.

Small N-terminal deletion by splicing in cerebellar alpha 6 subunit abolishes GABAA receptor function.

Sequence variation was found in cDNA coding for the extracellular domain of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit. About 20% of polymerase chain reaction (PCR)-amplified alpha 6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226-255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (alpha 6S) and long (wild-type) forms of the alpha 6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the alpha 6S subunit with the GABAA receptor beta 2 and gamma 2 subunits in human embryonic kidney 293 cells, were inactive at binding [3H]muscimol and [3H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by beta 2 gamma 2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the alpha 6 subunit-containing receptors.

Frequent loss of SMAD4/DPC4 protein in colorectal cancers.

BACKGROUND AND AIMS: Loss of DNA sequences from chromosome 18q21 is a major genetic change in colorectal tumorigenesis. Multiple genes have been identified in this area. One of these, DPC4 (deleted in pancreatic cancer 4, also known as SMAD4), is mutated in a minor subset of colorectal carcinomas as well as in germlines of humans predisposed to colon tumours. PATIENTS AND METHODS: The involvement of SMAD4 in sporadic colorectal neoplasia was evaluated by immunohistochemistry in 53 unselected cases and 27 cases displaying microsatellite instability. RESULTS: SMAD4 expression was absent in 20 of 53 (38%) unselected colorectal carcinomas, and reduced in another 15 (28%) cases. However, 26 of 27 cancers displaying microsatellite instability and TGF-betaIIR mutations were positive for SMAD4 immunostaining. CONCLUSIONS: Loss of SMAD4 expression may play a more prominent role in colon cancer than anticipated based on genetic evidence, but not in mutator phenotype tumours.

Molecular cloning and nucleotide sequence of chicken avidin-related genes 1-5.

Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1-5, avr1-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88-92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68-78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.

Mutation sharing, predominant involvement of the MLH1 gene and description of four novel mutations in hereditary nonpolyposis colorectal cancer. Mutations in brief no. 144. Online.

Worldwide, the DNA mismatch repair genes MSH2 and MLH1 account for a major share and almost equal proportions of hereditary nonpolyposis colorectal cancer (HNPCC). Furthermore, the predisposing mutation usually varies from kindred to kindred. In this study, we screen 29 verified or putative HNPCC kindreds from Finland for mutations in these two genes and found 8 different mutations, 7 in MLH1 and 1 in MSH2, occurring in 13 families. Four of these mutations were novel. Altogether, we have to date studied 81 kindreds for mutations and 12 different mutations in 52 families have been identified, 10 in MLH1 and 2 in MSH2. These data show that Finnish HNPCC kindreds are characterized by the predominant involvement of MLH1 (49/52, 94% of the families) and a high rate of shared mutations (5/12, 42%) offering unique possibilities for mutation screening for both research and diagnostic purposes.