Molecular characterization of bovine rotavirus strains circulating in northern Italy, 2003-2005.

A total of 232 stools collected from calves with rotavirus infection in herds located in northern Italy from 2003 to 2005 was investigated. Determination of the rotavirus G and P types was carried out using nested RT-PCR. G6 was the most prevalent genotype, accounting for 78.5% of samples, G10 accounted for 9.9% of samples and viruses of G8 type were found in 4.7% of samples. In 3% of samples, viruses were not classified due to concomitant infection with more G type strains, whereas viruses in 3.9% of samples could not be characterized with any of the G-specific primers used in this study. Most common P types were P[11] and P[5], accounting for 65.1% and 25%, respectively. In 2.6% of cases, samples reacted with multiple P-specific primers; no P[1] serotype was identified. The G6P[11] combination was predominant throughout the study period, i.e. 52.5% in 2003, 50% in 2004 and 40% in 2005. The incidence of G6P[5] increased from 13.1% in 2003 to 27% in 2004 and 25.5% in 2005. The G10P[11] combination decreased markedly from 18% in 2003 to 2.6% in 2004, rising again to 7.3% in 2005. G8P[11] viruses were similarly present in 2003 (5%) and 2004 (4.3%), declining slightly in 2005 (1.8%).

High prevalence of BRCA1 deletions in BRCAPRO-positive patients with high carrier probability.

Mutation screening of the BRCA1 and BRCA2 genes in probands with familial breast/ovarian cancer has been greatly improved by the multiplex ligation-dependent probe amplification (MLPA) assay able to evidence gene rearrangements not detectable by standard screening methods. However, no criteria for selection of cases to be submitted to the MLPA test have been reported yet. We used the BRCAPro software for the selection of familial breast/ovarian cancer probands investigated with the MLPA approach after negative BRCA1/2 conventional mutation screening. One hundred and seventy-seven probands were investigated for germline BRCA1/2 mutations after assessment of genetic risk using BRCAPro. Probands were classified as BRCAPro positive (n = 67) when the carrier probability (CP) was >10% and as BRCAPro negative (n = 110), when the CP was <10%. Conventional mutational analyses of the BRCA1/2 genes and, in one case, of p53 identified 22 pathogenetic germline mutations, 12 in BRCA1, 9 in BRCA2 and 1 in p53, in 22/177 (12.4%) probands. All the mutations except one were detected in BRCAPro-positive patients. In the 46 BRCAPro-positive cases that resulted negative by BRCA1/2 mutation, screening analysis of rearrangements within BRCA1/2 by MLPA was carried out. Three patients with a very high CP showed BRCA1 deletions, consisting of deletions of exons 1-2 in two probands and of exon 24 in the third proband. In one case, the exons 1-2 deletion was shown to cosegregate with disease in the family. No BRCA2 rearrangements were detected, but one patient showed the 1100delC of the CHEK2 gene, whose probe is present in the BRCA2 kit. In our series, the highest carrier detection rate of mutation screening plus MLPA analysis (52.3%) was in patients with a BRCAPro CP >50%.

Primary headaches in preschool age children: clinical study and follow-up in 163 patients.

Although headache frequency is increasing in preschool age children, an extensive evaluation of the clinical features in affected patients has yet to be achieved. This retrospective study examined 243 patients who were separately analysed in two distinct groups according to the age of onset and the age of first clinical evaluation. Group 1 included preschool age children, while Group 2 consisted of pubertal age patients. In all the patients the importance of a positive family history for headache as a risk factor was confirmed. In addition, when compared with Group 2, Group 1 showed greater male gender prevalence and earlier onset of the attacks. Regarding clinical features, in Group 1, compared with Group 2, the attack duration was shorter with lower symptom association such as photo- or phonophobia, nausea and no pain increase during physical activity. In the same group, tension-type headache was the predominant diagnosis, in contrast to the high migraine prevalence of Group 2. This study also showed that the International Headache Society 1988 classification criteria are not fully adequate for juvenile headaches. In fact, the headaches of more than 10% of patients in Group 1 still remained unclassifiable, while those of all the subjects in Group 2 were properly classifiable.

BRCA1 and BRCA2 mutations in breast/ovarian cancer patients from central Italy.

We report on the screening of the entire BRCA1/BRCA2 coding sequence by SSCP, PTT, and direct sequencing in 68 Italian families with recurrent breast or ovarian cancer. For each investigated proband, the probability of being carrier of a BRCA1/BRCA2 mutation was evaluated using the BRCAPRO software. We detected BRCA1/BRCA2 mutations in 8 patients (11.7%). However, if considering only patients with a carrier probability >10%, the detection rate was 36.8%, confirming the usefulness of the BRCAPRO software. One change (BRCA1 4172insT) was a novel mutation not reported in BIC database.

The Italian multi-centre project on evaluation of MRI and other imaging modalities in early detection of breast cancer in subjects at high genetic risk.

This report presents the preliminary results of the first phase (21 months) of a multi-centre, non-randomised, prospective study, aimed at evaluating the effectiveness of contrast-enhanced magnetic resonance imaging (MRI), X-ray mammography (XM) and ultrasound (US) in early diagnosis of breast cancer (BC) in subjects at high genetic risk. This Italian national trial (coordinated by the Istituto Superiore di Sanità, Rome) so far recruited 105 women (mean age 46.0 years; median age 51.0; age range 25-77 years), who were either proven BRCA1 or BRCA2 mutation carriers or had a 1 in 2 probability of being carriers (40/105 with a previous personal history of BC). Eight cases of breast carcinomas were detected in the trial (mean age 55.3 years, median age 52.5; age range 35-70 years; five with previous personal history of BC). All trial-detected BC cases (8/8) were identified by MRI, while XM and US correctly classified only one. MRI had one false positive case, XM and US none. Seven "MRI-only" detected cancers (4 invasive, 3 in situ) occurred in both pre- (n = 2) and post-menopausal (n = 5) women. With respect to the current XM screening programmes addressed to women in the age range 50-69 years, the global incidence of BC in the trial (7.6%) was over ten-fold higher. The cost per "MRI-only" detected cancer in this particular category of subjects at high genetic risk was substantially lower than that of an XM-detected cancer in the general women population. These preliminary results confirmed that MRI is a very useful tool to screen subjects at high genetic risk for breast carcinoma, not only in pre-, but also in post-menopausal age, with a low probability of false positive cases.

Thyroid carcinoma usually occurs in patients with familial adenomatous polyposis in the absence of biallelic inactivation of the adenomatous polyposis coli gene.

Papillary thyroid carcinoma (PTC) is a rare extracolonic manifestation of familial adenomatous polyposis, determined by germline mutations of the adenomatous polyposis coli (APC) gene. The aim of this study was to assess the presence of loss of heterozygosity of APC in the thyroid tumoral tissue. Specimens from six female patients, aged 20-36, were analyzed for germline and somatic mutations of the APC gene by restriction enzyme analysis and sequence analysis. Five of the six also had analysis for ret/PTC, a chimeric gene, the activation of which is restricted to papillary TC. Because a previous study showed that germline mutations in familial adenomatous polyposis-associated thyroid carcinoma were located between codons 140 and 1513, the search for somatic mutations of the APC gene was restricted to this genomic area. Three of the six patients, belonging to the same kindred, had a germline mutation at codon 1061. The remaining three, one per kindred, had germline mutations at codons 1061, 1061, and 1309, respectively. None of the six patients had loss of heterozygosity for APC or somatic mutation in the explored genomic area (codon 545 and codons 1061-1678). Four of five had activation of ret/PTC in the thyroid tumoral tissue, as ret/PTC1 isoform. Either APC has a tissue-specific dominant effect in the thyroid gland or the germline mutation confers a generic susceptibility to cancer development, but other factors (sex-related factors, environmental radiation, modifier genes) are also required for TC development. This usually involves ret/PTC activation, suggesting a possible cooperation between altered function of APC and gain of function of ret.

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.

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.

Unbalanced germ-line expression of hMLH1 and hMSH2 alleles in hereditary nonpolyposis colorectal cancer.

We analyzed the hMLH1 and hMSH2 genes in 30 unrelated hereditary nonpolyposis colorectal cancer (HNPCC) patients using mutational and immunohistochemical analyses combined whenever possible with primer extension assays, designed to estimate hMLH1 and hMSH2 transcript expression in peripheral blood lymphocytes. Single-strand conformational polymorphism screening and PCR-direct sequencing revealed seven hMLH1 and five hMSH2 sequence variants in 14 unrelated HNPCC patients, including three definite pathogenic mutations, four amino acid substitutions of uncertain pathogenic significance, and five polymorphisms. Immunohistochemistry indicated the lack of either hMLH1 or hMSH2 protein expression in tumors from 13 patients, and the absence of both hMLH1 and hMSH2 immunostaining was observed in the tumor from one additional case. The lack of hMLH1 or hMSH2 immunostaining was associated with the presence of microsatellite instability in the corresponding tumor and was also observed in tumors from patients negative for pathogenic mutations by mutational screening. There was a marked unbalance in the allelic expression of either hMLH1 or hMSH2 transcripts in three of eight unrelated HNPCC patients that could be analyzed, although a less marked unbalance was detected in two additional patients. Tumors from patients with germ-line unbalance in hMLH1 or hMSH2 transcript expression did not express the corresponding mismatch repair protein and displayed microsatellite instability. Our results indicate that constitutional alterations in hMLH1 and hMSH2 transcript expression may represent genetic markers for HNPCC carrier status also in cases in which mutational analysis did not detect a definite pathogenic variant. This suggests that transcript deregulation may represent a relevant mode of germ-line inactivation for mismatch repair genes.

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.

Origin and gender determination of dried blood on a statue of the Virgin Mary.

In Italy, blood exudation from objects of worship recurs frequently in ancient chronicles and literature, in popular beliefs, and even in modern mass-media reports. This phenomenon, that was associated with epochal or catastrophic events, has roots that reach classical antiquity. In the last few years, several events connected with the detection of bloody "tears" on statues of the Virgin Mary required forensic medicine investigations. In the present report we describe genetic investigations conducted on dried blood of unknown derivation found on a statuette representing the Virgin Mary. To test the human or animal origin of the blood, we amplified Alu-specific sequences from DNAs obtained from the unknown sample and from humans, large apes, various Old and New World monkeys, a prosimian, mouse, common domestic artiodactyls and chicken. This investigation restricted the range of possible origin of the statue blood to humans, apes and Old World monkeys. To test the male or female origin of the blood, we used a multiplex nested polymerase chain reaction method, that allows the simultaneous amplification of the X-specific locus DXZ4 and of the Y-specific locus SRY. Considering the unlikelihood of an origin from simian Old World primates, the exclusive amplification of the X-specific product from the unknown sample and from human female blood controls, compared to the amplification of distinct X- and Y-specific bands from human male blood controls, strongly supports a human female origin of the statue blood.

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.

Microsatellite instability is correlated with lymph node-positive breast cancer.

We analyzed 81 cases of primary breast carcinoma and 7 cases of fibroadenoma for microsatellite instability at eight loci. Twenty-seven cases (33.3%) manifested aberrant microsatellite alleles: 7 (8.6%) at one locus and 20 (24.7%) at two or more loci [tumors with replication error-positive (RER+) phenotype]. No evidence of microsatellite instability was observed in fibroadenomas. We investigated correlations between RER+ phenotype and clinicopathological characteristics of the carcinomas. The RER+ phenotype was statistically associated with large tumor diameter; of 19 RER+ tumors with measured size, 16 were > 2 cm, compared to 28 of 58 tumors with no evidence of microsatellite instability or with shifts in allele sizes limited to one locus (P

Microsatellite instability in early gastric cancer.

Microsatellite replication errors (RERs), consisting in random tumour-associated allele contractions or expansions, represent a frequent genetic alteration in gastric cancer and appear to be associated with important clinicopathologic parameters. To verify the role of microsatellite instability in the initial phases of gastric carcinogenesis, we analysed the status of II microsatellites in paired microdissected samples of tumour and unaffected mucosa from 30 cases of early gastric carcinoma. Fifteen tumours (50%) demonstrated RERs: these included 7 cases with RERs at one locus and 8 cases with RERs at 2 or more loci. Cases with 2 or more RERs were more frequent among intramucosal tumours, compared to tumours with submucosal spread (43% vs. 12%) and among tumours staged T1NOMx, compared to tumours staged T1N1Mx (35% vs. 0%). RER-positive microsatellite typings were statistically more frequent among tumours with intramucosal extension, lower stage (T1NOMx) and excavated growth pattern (macroscopic type III), compared to tumours with submucosal extension, higher stage (T1N1Mx) and elevated, flat or depressed growth patterns (macroscopic types IIa-IIb-IIc respectively). The above findings indicate that microsatellite instability occurs early in the progression of sporadic gastric cancer and tends to be associated with good prognostic indicators.

Congenital hypertrophy of the retinal pigment epithelium in familial adenomatous polyposis. Novel criteria of assessment and correlations with constitutional adenomatous polyposis coli gene mutations.

BACKGROUND: Congenital hypertrophy of the retinal pigment epithelium (CHRPE) is the most common extracolonic manifestation of familial adenomatous polyposis (FAP) and is an early clinical marker of the disease. It seems to be correlated with the position of constitutional mutations of the adenomatous polyposis coli (APC) gene. METHODS: The authors investigated the expression of CHRPE and its correlation with the position of the APC gene in FAP patients and in "at risk" relatives from 31 FAP kindreds. To obtain comparable data on CHRPE expression, the authors developed a novel scoring system based on morphologic and dimensional criteria. RESULTS: A positive CHRPE score was obtained in 29 of 39 FAP patients (74%) and in 16 of 53 relatives who showed no clinical evidence of FAP (30%). Colonoscopy revealed polyps in 20 of the 47 relatives who could be examined. The cumulative sensitivity and specificity of CHRPE were 72.88% and 96.29%, respectively. APC gene mutations were characterized in 34 subjects from 17 kindreds. In 28 of the subjects, mutations were detected in exon 15, between codons 876 and 1324. Mutations were found in exon 9 in 6 subjects. In 3 of the 6 subjects, they were found at the site where both forms of alternative splicing of the exon occur (codon 437). In the other 3 subjects (another kindred), mutations were found in the portion of exon 9 in which alternative splicing occurs (codon 367). Only 1 of the 6 subjects (16.6%) with mutations in exon 9 had a positive CHRPE score, compared with 28 of 28 subjects (100%) with mutations in exon 15. None of the 3 subjects with mutations in codon 437 had a positive CHRPE score. The CHRPE scores of exon 15 mutation carriers varied markedly both within and among kindreds, irrespective of the mutation site. CONCLUSIONS: The results of this study indicate that the site of APC gene mutation influences CHRPE expression but is not the only factor responsible for the presence and level of retinal lesions in FAP patients.