The type of K-ras mutation determines prognosis in colorectal cancer.

BACKGROUND: Mutations involving the oncogene K-ras in colorectal cancer may be related to tumor aggressiveness. However, the value of K-ras gene determination as a prognostic marker has not been clearly established. PATIENTS AND METHODS: The results from 98 patients recruited in a prospective study analyzing the effect of a K-ras mutation as a prognostic factor in colorectal cancer are reported. RESULTS: Disease-free (P = 0.02) and overall survival (P = 0.03) were significantly reduced for patients harboring a K-ras mutation. Two specific mutations demonstrated a significantly increased risk of disease recurrence, namely, 12-TGT (P = 0.04) and 13-GAC substitutions (P = 0.002). Patients with either of these substitutions had a 2-year disease-free survival rate of 37% compared with that of 67% for the group of patients harboring any other mutation type or a wild-type status (P = 0.01). CONCLUSIONS: The results herein presented suggest that K-ras acts as a prognostic factor in colorectal cancer and that this effect is probably related to a limited number of defined mutations.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.

Allelic variation in the VMD2 gene in best disease and age-related macular degeneration.

PURPOSE: To assess the allelic variation of the VMD2 gene in patients with Best disease and age-related macular degeneration (AMD). METHODS: Three hundred twenty-one AMD patients, 192 ethnically similar control subjects, 39 unrelated probands with familial Best disease, and 57 unrelated probands with the ophthalmoscopic findings of Best disease but no family history were screened for sequence variations in the VMD2 gene by single-strand conformation polymorphism (SSCP) analysis. Amplimers showing a bandshift were reamplified and sequenced bidirectionally. In addition, the coding regions of the VMD2 gene were completely sequenced in six probands with familial Best disease who showed no SSCP shift. RESULTS: Forty different probable or possible disease-causing mutations were found in one or more Best disease or AMD patients. Twenty-nine of these variations are novel. Of the 39 probands with familial Best disease, mutations were detected in all 39 (33 by SSCP and 6 by DNA sequencing). SSCP screening of the 57 probands with a clinical diagnosis of Best disease but no family history revealed 16 with mutations. Mutations were found in 5 of 321 AMD patients (1.5%), a fraction that was not significantly greater than in control individuals (0/192, 0%). CONCLUSIONS: Patients with the clinical diagnosis of Best disease are significantly more likely to have a mutation in the VMD2 gene if they also have a positive family history. These findings suggest that a small fraction of patients with the clinical diagnosis of AMD may actually have a late-onset variant of Best disease, whereas at the same time, a considerable fraction of isolated patients with the ophthalmoscopic features of Best disease are probably affected with some other macular disease.

Identification of a novel splice-site mutation in the CYP1A2 gene.

AIMS: To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. METHODS: The nucleotide sequence of the 7 exons, exon-intron boundaries and 5'-flanking region of the CYP1A2 gene was analysed by direct sequencing. RESULTS: Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. CONCLUSIONS: This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.

Mutation hot spots in 5q31-linked corneal dystrophies.

Mutations in the BIGH3 gene on chromosome 5q31 cause four distinct autosomal dominant diseases of the human cornea: granular (Groenouw type I), Reis-Bücklers, lattice type I, and Avellino corneal dystrophies. All four diseases are characterized by both progressive accumulation of corneal deposits and eventual loss of vision. We have identified a specific recurrent missense mutation for each type of dystrophy, in 10 independently ascertained families. Genotype analysis with microsatellite markers surrounding the BIGH3 locus was performed in these 10 families and in 5 families reported previously. The affected haplotype could be determined in 10 of the 15 families and was different in each family. These data indicate that R555W, R124C, and R124H mutations occurred independently in several ethnic groups and that these mutations do not reflect a putative founder effect. Furthermore, this study confirms the specific importance of the R124 and R555 amino acids in the pathogenesis of autosomal dominant corneal dystrophies linked to 5q.

p53 and Ki-ras as prognostic factors for Dukes' stage B colorectal cancer.

Mutations of the TP53 and Ki-ras genes have been reported to be of prognostic importance in colorectal carcinomas. An increased intracellular concentration of the p53 protein, although not identical to, is sometimes seen in tumours with TP53 mutation and has been correlated with poor prognosis in some tumour types. Previous colorectal cancer studies, addressing the prognostic importance of Ki-ras mutation and TP53 aberrations, yielded contradictory results. The aim of this study was to determine in a clinically and therapeutically homogeneous group of 122 sporadic Dukes' B colorectal carcinomas with a median follow-up of 67 months (3-144 months) whether or not p53 protein expression, TP53 mutation and K-ras mutation correlated with prognosis. p53 staining was performed by immunohistochemistry, using the monoclonal antibody DO7 on paraffin-embedded tissue. Mutations in exons 5-8 of the TP53 gene and in codons 12 and 13 of the K-ras gene were assayed in paraffin-embedded tissue by the single-strand conformation polymorphism (SSCP) assay. Nuclear p53 staining was found in 57 (47%) tumours. Aberrant migration patterns indicating mutation of the TP53 gene were found in 39 (32%) tumours. Forty-six carcinomas (38%) showed a mutation of the Ki-ras codons 12 or 13. In a univariate analysis, patients with wild-type TP53 status showed a trend towards better survival, compared with those with mutated TP53 (log-rank test, P = 0.051). Likewise, tumours immunohistochemically positive for p53 showed a worse prognosis than p53-negative tumours (P = 0.010). The presence or absence of mutations in Ki-ras did not correlate with prognosis (P = 0.703). In multivariate analysis, only p53 immunoreactivity emerged as an independent marker for prognosis hazard ratio (HR) = 2.16, 95% confidence interval (CI) 1.12-4.11, P = 0.02). Assessment of p53 protein expression is more discriminative than TP53 mutation to predict the outcome of Dukes' stage B tumours and could be a useful tool to identify patients who might benefit from adjuvant therapy.

Low Y chromosome variation in Saudi-Arabian hamadryas baboons (Papio hamadryas hamadryas).

It is important to characterise the amount of variation on the mammalian Y chromosome in order to assess its potential for use in evolutionary studies. We report very low levels of polymorphism on the Y chromosome of Saudi-Arabian hamadryas baboons, Papio hamadryas hamadryas. We found no segregating sites on the Y, despite sequence analysis of 3 kb noncontiguous intron sequence in 16 males with divergent autosomal microsatellite genotypes, and a further analysis of 1.1 kb intron sequence in 97 males from four populations by SSCP. In addition, we tested seven human-derived Y-linked microsatellites in baboons. Only four of these loci were male-specific and only one was polymorphic in our 97 male sample set. Polymorphism on the Y chromosome of Arabian hamadryas appears to be low compared to other primate species for which data are available (eg humans, chimpanzees and bonobos). Low effective population size (Ne) of paternal genes due to polygyny and female-biased adult sex ratio is a potential reason for low Y chromosome variation in this species. However, low Ne for the Y should be counterbalanced to some extent by the species' atypical pattern of male philopatry and female-biased dispersal. Allelic richness averaged over seven loci was not significantly different between an African and an Arabian population, suggesting that loss of variation during the colonisation of Arabia does not explain low Y variation. Finally, in the absence of nucleotide polymorphism, it is unclear to what extent selection could be responsible for low Y variation in this species.

No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Evidence for an association with type 2 diabetes mellitus at the PPARG locus in a South Indian population

Peroxisome proliferator-activated receptor-gamma2 (PPARG2) is a nuclear hormone receptor of ligand-dependent transcription factor involved in adipogenesis and a molecular target of the insulin sensitizers thiazolidinediones. We addressed the question of whether the 3 variants (-1279G/A, Pro12Ala, and His478His) in the PPARG2 gene are associated with type 2 diabetes mellitus and its related traits in a South Indian population. The study subjects (1000 type 2 diabetes mellitus and 1000 normal-glucose-tolerant subjects) were chosen randomly from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The variants were screened by single-stranded conformational variant, direct sequencing, and restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. The -1279G/A, Pro12Ala, and His478His variants of the PPARG2 gene were not associated with type 2 diabetes mellitus. However, the 2-loci analyses showed that, in the presence of Pro/Pro genotype of the Pro12Ala variant, the -1279G/A promoter variant showed increased susceptibility to type 2 diabetes mellitus (odds ratio, 2.092; 95% confidence interval, 1.22-3.59; P = .008), whereas in the presence of 12Ala allele, the -1279G/A showed a protective effect against type 2 diabetes mellitus (odds ratio, 0.270; 95% confidence interval, 0.15-0.49; P < .0001). The 3-loci haplotype analysis showed that the A-Ala-T (-1279G/A-Pro12Ala-His478His) haplotype was associated with a reduced risk of type 2 diabetes mellitus (P < .0001). Although our data indicate that the PPARG2 gene variants, independently, have no association with type 2 diabetes mellitus, the 2-loci genotype analysis involving -1279G/A and Pro12Ala variants and the 3-loci haplotype analysis have shown a significant association with type 2 diabetes mellitus in this South Indian population.

Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.

Genetic uniformity of international isolates of Leifsonia xyli subsp xyli, causal agent of ratoon stunting disease of sugarcane

An international collection of the sugarcane ratoon stunting disease pathogen, Leifsonia xyli subsp. xyli, was analysed to assess genetic diversity. DNA fingerprinting using BOX primers was performed on 105 isolates, comprising 65 Australian isolates and an additional 40 isolates from Indonesia (n = 8), Japan (n = 1), USA (n = 3), Brazil (n = 2), Mali (n = 2), Zimbabwe (n = 13), South Africa (n = 9) and Reunion (n = 2). Sixty-two of these isolates were also screened using ERIC primers. No variation was found among any of the isolates. The intergenic spacer (IGS) region of the ribosomal RNA genes from 54 isolates was screened for sequence variation using single-stranded conformational polymorphism (SSCP), but none was observed. Direct sequencing of the IGS from a subset of nine isolates, representing all of the countries sampled in this study, confirmed the results of the SSCP analysis. Likewise, no sequence variation was found in the 16S ribosomal RNA genes of the same subset. Four Colombian isolates from sugarcane, morphologically similar to L. xyli subsp. xyli, were putatively shown to be an undescribed Agrococcus species of unknown pathogenicity. The lack of genetic variation among L. xyli subsp. xyli isolates, independent of time of sampling, cultivar of isolation, or country of origin, suggests the worldwide spread of a single pathogenic clone, and further suggests that sugarcane cultivars resistant to ratoon stunting disease in one area should retain this property in other regions.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsα and Gi2α genes

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsα, Gi2α and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsα, and Gi2α contribute little if any to cranipharyngioma development.

Conformational changes modulate the activity of human RAD51 protein.

Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homologue, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity.

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.