Searching for the best real-time RT-PCRs to detect Zika virus infections: the importance of comparing several protocols.

Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.

Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2.

BACKGROUND AND AIMS: Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play an important role in intestinal epithelium homeostasis. Insulin receptor substrate 2 (IRS2) is a poorly characterised component in this pathway. METHODS: Using complementary in vitro and in vivo human and murine models, expression (mRNA and protein levels), localisation (immunohistochemistry) and regulation of IRS2 were investigated in the normal intestine and colorectal tumours. In silico analysis of the human IRS2 promoter was performed together with reporter and chromatin immunoprecipitation assays. RESULTS: Significant IRS2 expression was detected in the intestine, with specific protein localisation in the villus region of the ileum and in the surface epithelium of the colon. In human HT29 and Caco2 cells, IRS2 mRNA levels increased with spontaneous and induced differentiation, together with CDX2 (caudal-related homeobox protein 2), P21 and KLF4 (Krüppel-like factor 4). Adenoviral infection with human CDX2 induced IRS2 expression in APC- (adenomatous polyposis coli) and beta-catenin-mutated cells. On the other hand, IRS2 downregulation was observed in differentiated enterocytes after adenoviral infection with short hairpin CDX2 (shCDX2), in the intestine of CDX2 heterozygous mice and in colorectal tumours of Apc(Min/+) and patients with familial adenomatous polyposis (FAP). The human IRS2 promoter region presents several CDX2-binding sites where CDX2 immunoprecipitated in vivo. IRS2 reporters were functionally activated via CDX2 and blocked via a dominant-negative CDX2 protein. CONCLUSIONS: Combining gain- and loss-of-function approaches, an intriguing scenario is presented whereby IRS2 is significantly expressed in the apical intestinal compartment and is directly controlled by CDX2 in normal intestine and tumours.

Patterns of K-ras mutation in colorectal carcinomas from Iran and Italy (a Gruppo Oncologico dell'Italia Meridionale study): influence of microsatellite instability status and country of origin.

BACKGROUND: K-ras mutations are a key step in colorectal cancer progression. Such mutations have been widely studied in case series from Western countries but there are few data on the rate and spectrum of mutations in tumors from countries where the epidemiological features of the disease are different. PATIENTS AND METHODS: Tumor samples from 182 Iranian colorectal cancer patients (170 sporadic cases and 12 HNPCC cases) were screened for K-ras mutations at codons 12, 13 and 61 by sequencing analysis. The cases were also characterized for microsatellite instability at mononucleotide repeats by PCR and fragment analysis, and classified according to microsatellite instability status. The frequency and the spectrum of K-ras mutations were compared with those observed in a series of colorectal cancer patients from Italy. RESULTS: K-ras mutations were observed in 68/182 (37.4%) cases. Mutation frequencies were similar in HNPCC-associated, sporadic MSI-H and sporadic microsatellite-stable (MSS) tumors. However, the G13D substitution was more frequent in HNPCC (3/4, 75%) and sporadic MSI-H (7/11, 63.6%) tumors compared to sporadic MSS tumors (11/53, 20.4%) (P <0.01). Comparison of mutations in the two series from Iran and Italy showed a significantly higher frequency of G13D among Italian patients. CONCLUSIONS: While the frequency of K-ras mutations could be similar, the mutational spectrum could be differentially influenced by genetic and environmental factors.

Tyr(612) and Tyr(632) in human insulin receptor substrate-1 are important for full activation of insulin-stimulated phosphatidylinositol 3-kinase activity and translocation of GLUT4 in adipose cells.

To examine contributions of specific YXXM motifs in human insulin receptor substrate-1 (IRS-1) to mediating the metabolic actions of insulin, we studied IRS-1 mutants containing various substitutions of Phe for Tyr. In transfected NIH-3T3(IR) cells, insulin stimulation caused a 5-fold increase in phosphatidylinositol 3-kinase (PI3K) activity coimmunoprecipitated with wild-type IRS-1. No PI3K activity was associated with IRS1-F6 (Phe substituted for Tyr at positions 465, 612, 632, 662, 941, and 989). Adding back both Tyr(612) and Tyr(632) fully restored IRS-1-associated PI3K activity, whereas adding back either Tyr(612) or Tyr(632) alone was associated with intermediate PI3K activity. In rat adipose cells transfected with epitope-tagged GLUT4, insulin stimulation caused a 2-fold increase in cell surface GLUT4-HA. Cotransfection of cells with GLUT4-HA and either wild-type IRS-1 or IRS1-Y612/Y632 increased basal cell surface GLUT4-HA (in the absence of insulin) to approximately 80% of the levels seen in insulin-stimulated control cells, whereas overexpression of IRS1-F6 had no effect on the insulin dose-response curve. Overexpression of IRS1-Y612 or IRS1-Y632 caused intermediate effects. Thus, both Tyr(612) and Tyr(632) are important for IRS-1 to fully activate PI3K and mediate translocation of GLUT4 in response to insulin.

Protein kinase C-zeta phosphorylates insulin receptor substrate-1 and impairs its ability to activate phosphatidylinositol 3-kinase in response to insulin.

Protein kinase C-zeta (PKC-zeta) is a serine/threonine kinase downstream from phosphatidylinositol 3-kinase in insulin signaling pathways. However, specific substrates for PKC-zeta that participate in the biological actions of insulin have not been reported. In the present study, we identified insulin receptor substrate-1 (IRS-1) as a novel substrate for PKC-zeta. Under in vitro conditions, wild-type PKC-zeta (but not kinase-deficient mutant PKC-zeta) significantly phosphorylated IRS-1. This phosphorylation was reversed by treatment with the serine-specific phosphatase, protein phosphatase 2A. In addition, the overexpression of PKC-zeta in NIH-3T3(IR) cells caused significant phosphorylation of cotransfected IRS-1 as demonstrated by [(32)P]orthophosphate labeling experiments. In rat adipose cells, endogenous IRS-1 coimmunoprecipitated with endogenous PKC-zeta, and this association was increased 2-fold upon insulin stimulation. Furthermore, the overexpression of PKC-zeta in NIH-3T3(IR) cells significantly impaired insulin-stimulated tyrosine phosphorylation of cotransfected IRS-1. Importantly, this was accompanied by impaired IRS-1-associated phosphatidylinositol 3-kinase activity. Taken together, our results raise the possibility that IRS-1 is a novel physiological substrate for PKC-zeta. Because PKC-zeta is located downstream from IRS-1 and phosphatidylinositol 3-kinase in established insulin signaling pathways, PKC-zeta may participate in negative feedback pathways to IRS-1 similar to those described previously for Akt and GSK-3.

Interaction between the G1057D variant of IRS-2 and overweight in the pathogenesis of type 2 diabetes.

The insulin receptor substrate-2 (IRS-2) is a major insulin signalling molecule. IRS-2 inactivation in mice induces a form of diabetes characterized by peripheral insulin resistance and reduced beta cell mass. We tested the hypothesis that a common non-conservative amino acid substitution of IRS-2 (G1057D) might interact with overweight in the pathogenesis of type 2 diabetes. The variant was genotyped in 193 Italian patients with type 2 diabetes and 206 control subjects. In the absence of overweight, the risk of type 2 diabetes decreased according to the dosage of the D1057 allele (odds ratio for GD genotype 0.46 [95% CI 0.25-0.86]; DD genotype 0.18 [0.04-0.68]; P for trend = 0.0012). Conversely, the interaction between overweight and genotype increased the risk of type 2 diabetes according to the dosage of the D1057 allele (odds ratio for GD genotype 2.50 [1.11-5.65]; DD genotype 5.74 [1.11-29. 78]; P for trend = 0.0047). Among controls, fasting C-peptide levels, after adjustment for plasma glucose, were inversely related to the dosage of the D1057 allele (P = 0.020). This finding suggested that carriers of the D1057 allele may have higher insulin sensitivity and supported the protective effect of this allele. Conversely, among overweight patients there was a parallel increase in fasting plasma glucose (P for trend = 0.037) and fasting C-peptide according to the dosage of the D1057 allele, suggesting that higher insulin resistance and relative beta cell failure contributed to the increased risk of type 2 diabetes in overweight carriers of this allele. These data provide evidence for a strong association between type 2 diabetes and the G1057D common genetic variant of IRS-2, which appears to be protective against type 2 diabetes in a codominant fashion. Overweight appears to modify the effect of this polymorphism toward a higher risk of type 2 diabetes. Carriers of this polymorphism may represent an elective target for prevention of type 2 diabetes through preventing or treating excessive weight.

Different subcellular localization and phosphoinositides binding of insulin receptor substrate protein pleckstrin homology domains.

Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.

Mutational analysis of OB gene in obese and type 2 diabetes affected subjects.

Peripheral blood DNA from 12 subjects affected by familial obesity and from 35 subjects affected by type 2 diabetes were analysed for mutations in the coding sequence of the OB gene. Mutational analysis, conducted using the single strand conformation polymorphism (SSCP) technique, followed by direct sequencing did not reveal the presence of nucleotide variants in the coding region of the OB gene. The lack of mutations in the coding sequence is consistent with previous data suggesting that mutations in the coding sequence of the OB gene are not common in human familial obesity. In 2 samples displaying a non-informative pattern of SSCP and in 8 additional samples the nucleotide sequence of portion of the intron 2 bordering the coding sequence of exon 2 identified a G in the positions +14IVS and +18IVS, according to a sequence reported previously, but in contrast with some others. All samples were homozygous for these intron variants.

APC gene mutations and colorectal adenomatosis in familial adenomatous polyposis.

Correlations between germline APC mutation sites and colorectal pathophenotypes, as evaluated by the direct count of adenomas at colectomy, were investigated analysing colectomy specimens from 29 FAP patients carrying one mis-sense (codon 208) and 14 frame-shift or non-sense APC mutations (codons 232, 367, 437, 623, 876, 995, 1061, 1068, 1075, 1112, 1114, 1309, 1324, 1556). The mis-sense mutation at codon 208 was associated with a relatively mild colorectal pathophenotype. The mutation at codon 367, subject to alternative splicing, was associated with attenuated FAP. The mutation at codon 1309 was associated with the profuse colorectal adenomatosis. For 13 mutations, predicted to result in null alleles or truncated APC proteins, we correlated density and distribution of colorectal adenomas with the predicted functional effects of the mutation. The most severe colorectal pathophenotype was significantly associated with the truncating mutation at codon 1309, which is located downstream to the I beta-catenin binding domain but upstream II beta-catenin-binding domain. Mutations between codons 867 and 1114, which affect the I beta-catenin binding domain, as well as mutations occurring in exons 6 and 9, predicted to result in null alleles, were associated with a less severe colorectal pathophenotype. Overall, the highest number of adenomas was detected in the right colon, followed by the left colon, transverse colon sigma and rectum. However, the highest density of adenomas was observed in the left colon, followed by the right colon, sigma, transverse colon and rectum. Colorectal carcinomas, observed in only five patients, were all in the left colon.

Transcripts with splicings of exons 15 and 16 of the hMLH1 gene in normal lymphocytes: implications in RNA-based mutation screening of hereditary non-polyposis colorectal cancer.

Germline mutations of the hMLH1 gene are estimated to account for a large fraction of kindreds affected by hereditary non-polyposis colorectal cancer (HNPCC). In a significant number of cases, hMLH1 mutations result in the expression of truncated proteins. We report here two novel alternatively spliced forms of hMLH1 mRNA in normal lymphocytes. One of these novel isoforms lacks the coding region of the gene between codons 557 and 578, corresponding to the entire exon 15. The deletion introduces a frameshift that results in a premature stop signal. The other isoform is characterised by an in-frame deletion spanning codons 578-632, corresponding to loss of the entire exon 16. Further studies are necessary to establish the biological significance of these alternative splicings. The presence of alternatively spliced hMLH1 transcripts that mimic pathogenic mutations should be taken into account in the mutational screening of the hMLH1 gene by reverse transcription-polymerase chain reaction methodologies.

Optimized PCR labeling in mutational and microsatellite analysis.

To optimize the labeling and visualization of PCR products we tested different variables, including deoxynucleotide concentration and ratio, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols. Labeling was achieved using a fixed amount of labeled dATP, whose relative specific activity was varied by adding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 micromol/L, with a peak at 1.8 micromol/L. This concentration corresponded to an optimal ratio between the increase in specific activity and the decrease in DNA yield. Nucleotide imbalances >1:2 were not advantageous. Mutational analysis by single-strand conformational polymorphism (SSCP) was used to validate PCR-labeling protocols. The limiting nucleotide concentrations did not affect SSCP. Clear SSCP patterns were obtained using DNA templates of different sizes derived from several genes. SSCP patterns obtained using one-step or nested PCR-labeling protocols were equivalent and were visualized after overnight exposure, using [alpha35S]dATP as the label. Dilutions of labeled products ranging between 1:10 and 1:2.5 influenced SSCP patterns, and the lowest dilution tested produced better-defined and more-intense signals. Optimized SSCP conditions allowed the detection of novel and previously characterized nucleotide variants. Clear microsatellite typing was also obtained using optimized protocols and [alpha35S]dATP as the label.

Transcript dosage effect in familial adenomatous polyposis: model offered by two kindreds with exon 9 APC gene mutations.

Analysis of genotype-phenotype correlations in familial adenomatous polyposis (FAP) patients demonstrated that the phenotypic heterogeneity of FAP is partly related to the mutation site. We investigated the molecular basis for the difference in severity of colorectal disease observed comparing FAP patients from two kindreds with neighbouring germline mutations in exon 9 of the APC gene. Patients from one kindred presented with a attenuated form of FAP, characterized by a low number of colorectal adenomas (up to 22). In FAP patients from this kindred, the APC gene mutation was localized at codon 367, in the portion of exon 9 that is alternately spliced. This is expected to result in the splicing-out of the mutation site in a fraction of mRNA molecules and in the residual production of wild-type transcripts from the mutant APC allele. Patients from the other kindred manifested a FAP phenotype characterized by hundreds of colorectal adenomas (320 to > 500). In these patients, the APC gene mutation abolished the donor site of exon 9a, used in both alternately spliced isoforms of the exon. The analysis of the relative levels of mutant and wild-type transcripts in unaffected colonic mucosa demonstrated that the mutant allele was not expressed. The model offered by our FAP patients with neighbouring exon 9 APC mutations supports the view that in addition to the mutation site, the type of mutation and transcript dosage effects contribute to the heterogeneity of disease phenotypes.

Novel allele of the insulin receptor substrate-1 bearing two non-conservative amino acid substitutions in a patient with noninsulin-dependent diabetes mellitus. Mutations in brief no. 130. Online.

We analyzed by SSCP the complete IRS-1 coding sequence in NIDDM patient #25 D. Unique conformers corresponding to a Ser to Tyr substitution at codon 1043 (S1043Y), and to a Cys to Tyr substitution at codon 1095 (C1095Y) were detected in this patient. The results of sequential digestion with restriction enzymes indicated that the novel sequence variants segregate on the same allele. Relatives of patient #25 D were not available for study, to confirm segregation of the novel allele with NIDDM in the family. Several lines of evidence suggest that the non-conservative amino acid substitutions detected in NIDDM patient #25 D have the potential to affect IRS-1 functions and could play a pathogenic role in this patient. Both S1043Y and C1095Y occur in a highly conserved sequence from human skeletal muscle, human hepatoma, mouse, and rat IRS-1. Protein subsequence analysis revealed that the S1043Y substitution abolishes a consensus sequence for glycogen synthase kinase 3 phosphorylation. Furthermore, S1043Y and C1095Y are not common IRS-1 polymorphisms as they were detected only in 1/136 choromosomes from NIDDM patients (allele frequency in NIDDM patients = 0.0007) and in 0/120 chromosomes from control subjects.

Tyrosine residues in the C-terminal domain of the insulin-like growth factor-I receptor mediate mitogenic and tumorigenic signals.

We investigated cellular proliferation, the transforming activity, and activation of known signal transduction pathways in NIH-3T3 cells stably expressing insulin-like growth factor-I receptors (IGF-IRs) with amino acid substitutions in the carboxy(C)-terminal domain. The mutant receptors contained substitutions of both tyrosines 1250 and 1251 with phenylalanine and histidine (amino acids present in the analogous positions in the insulin receptor), as well as phenylalanine 1310 replaced by tyrosine (IsY clones) to resemble the placement of tyrosine residues in the C-terminal domain of the insulin receptor. As a control for the IsY clones, a second mutant receptor was expressed with a substitution of phenylalanine 1310 with tyrosine only (DBY clones). Clones expressing IGF-IRs with the IsY substitutions had a significantly slower rate of growth compared with cells expressing an equivalent number of wild-type IGF-IRs (NWT). In contrast, the DBY clones showed relatively normal growth rates. Cells with wild-type IGF-IR demonstrated a transformed phenotype in soft agar assays. The IsY clones lost the transforming ability of the wild type IGF-IR, whereas DBY clones formed colonies. IGF-I-stimulated autophosphorylation of the IGF-IR and tyrosine phosphorylation of IRS-1 and SHC, known substrates in the IGF-IR signal transduction pathway, were studied. Mutated IGF-IRs (IsY and DBY) did not alter the IGF-I-induced tyrosine phosphorylation of these proteins. Furthermore, the mutated IGF-IRs did not alter Grb2 association with phosphorylated IRS-1 and SHC. IGF-I stimulation of Crk-II phosphorylation, a novel substrate of the IGF-IR, was similar in cells expressing mutated and wild-type IGF-IRs. IGF-I-induced activation of phosphatidylinositol (PI) 3'-kinase was equivalent in cells expressing either mutant or wild-type IGF-IRs. These data suggest that the IGF-IR mediates, at least in part, cellular proliferation and increased transforming ability through its C-terminal domain. The exact postreceptor signaling pathway(s) involved have yet to be fully elucidated.

Alterations of microsatellites in neurofibromas of von Recklinghausen's disease.

von Recklinghausen's disease, or type I neurofibromatosis, a common familial tumor syndrome, is characterized by the occurrence of multiple benign neoplasms of nerve sheath cells. The disease is caused by germ-line mutations of the NF1 gene, which encodes a member of the GTPase-activating superfamily of Ras regulatory proteins. We analyzed 5 dinucleotide repeat loci in DNAs from neurofibromas and matched normal skin from 16 NF1 patients. Eight cases (50%) manifested microsatellite alterations. Expansions or compressions of dinucleotide repeats were observed at one locus in four cases and at two loci in one case. Banding patterns compatible with the loss of a microsatellite allele were observed in four cases, including one that also presented microsatellite instability. The surprisingly high frequency of microsatellite alterations suggests that the NF1 gene or another gene(s) contributing to the pathogenesis of neurofibromas might be directly or indirectly implicated in the control of genomic integrity.

Multiplex PCR analysis and genotype-phenotype correlations of frequent APC mutations.

Germline mutations of the adenomatous polyposis coli (APC) gene tend to cluster in discrete regions. Some of these mutations occur frequently in familial adenomatous polyposis coli (FAP) patients, and strategies for genetic diagnosis of the disease should include simple methods for their detection. We studied a total of 48 FAP-affected or "at-risk" members from 31 unrelated FAP pedigrees. Unrelated patients were analyzed using heteroduplex analysis on agarose minigels (HAAM) and multiplex allele-specific PCR. This novel strategy readily and reliably detected the three frequently occurring APC deletions at codons 1061, 1068, and 1309, allowing identification of mutant alleles in nine unrelated patients. A targeted mutational analysis, based on HAAM and amplification refractory mutation system (ARMS), allowed the rapid identification of 11 additional subjects with germline deletions, among relatives of the patients in whom mutations had been detected by multiplex PCR and HAAM. The use of two independent PCR-based tests, employing distinct sets of primers, reduces the possibility that artifacts occurring during DNA amplification may interfere with the diagnostic evaluation. The analysis of genotype-phenotype correlations provided evidence for heterogeneity with regard to the extent of colonic and extracolonic manifestations of the disease in subjects bearing identical mutations. However, the consistent association of the deletion at codon 1309 with more severe colonic disease than that observed in patients with mutations at codons 1061 and 1068, supports a correlation between mutation site and penetrance of FAP.

p53 expression and genetic evidence for viral infection in intraepithelial neoplasia of the uterine cervix.

Infections with high-risk strains of human papillomaviruses (HPVs) and with herpes simplex virus type 2 (HSV 2), as well as inactivation of the p53 tumor suppressor gene, are important cofactors in cervical carcinogenesis. We analyzed 41 paraffin-embedded cervical intraepithelial lesions, including 25 cases of low-grade cervical intraepithelia neoplasia (CIN), and 16 cases of high-grade CIN for the presence of HPV 16/18 and HSV 2 genomic sequences and for the nuclear accumulation of the p53 protein. HPV 16 DNA was detected in 24.% of low-grade CINs and in 43.7% of high-grade CINs. HPV 18 was found only in 8.% of low-grade CINs. None of the cases tested scored positive for HSV 2 DNA. Nuclear accumulation of p53 was found in 4% of low-grade CINs, and in 31.2% of high-grade CINs, including 57.1% of the lesions that were positive for HPV 16. These results indicate that HPV 16 infection was over sixfold more common than HPV 18 infection and that p53 overexpression was significantly associated with high-grade lesions.