Analysis of telomere dynamics in peripheral blood cells from patients with Lynch syndrome.

BACKGROUND: In patients with Lynch syndrome, germline mutations in DNA mismatch repair (MMR) genes cause a high risk of developing a broad spectrum of cancers. To date, the management of patients with Lynch syndrome has represented a major challenge because of large variations in age at cancer onset. Several factors, including genetic anticipation, have been proposed to explain this phenotypic heterogeneity, but the molecular mechanisms remain unknown. Telomere shortening is a common event in tumorigenesis and also has been observed in different familial cancers. In this study, the authors investigated the possibility of a relation between telomere length and cancer onset in patients with Lynch syndrome. METHODS: The mean telomere length was measured using quantitative polymerase chain reaction in peripheral blood samples from a control group of 50 individuals, from 31 unaffected mutation carriers, and from 43 affected patients, and the results were correlated with both gene mutation and cancer occurrence. In affected patients, telomere attrition was correlated with age at cancer onset. In all patients, a t test was used to assess the linearity of the regression. RESULTS: A significant correlation between telomere length and age was observed in both affected and unaffected mutation carriers (P = .0016 and P = .004, respectively) and in mutS homolog 2 (MSH2) mutation carriers (P = .0002) but not in mutL homolog 1 (MLH1) mutation carriers. Telomere attrition was correlated significantly with age at onset in MSH2 carriers (P = .004), whereas an opposite trend toward longer telomeres in patients with delayed onset was observed in MLH1 carriers. CONCLUSIONS: The current data suggested that telomere dynamics differ between MLH1 and MSH2 mutation carriers. It is possible that subtle, gene-specific mechanisms can be linked to cancer onset and anticipation in patients with Lynch syndrome.

Breakpoint determination of 15 large deletions in Peutz-Jeghers subjects.

The Peutz-Jeghers Syndrome (PJS) is an autosomal dominant polyposis disorder with increased risk of multiple cancers. STK11/LKB1 (hereafter named STK11) germline mutations account for the large majority of PJS cases whereas large deletions account for about 30% of the cases. We report here the first thorough molecular characterization of 15 large deletions identified in a cohort of 51 clinically well-characterized PJS patients. The deletions were identified by MLPA analysis and characterized by custom CGH-array and quantitative PCR to define their boundaries. The deletions, ranging from 2.9 to 180 kb, removed one or more loci contiguous to the STK11 gene in six patients, while partial STK11 gene deletions were present in the remaining nine cases. By means of DNA sequencing, we were able to precisely characterize the breakpoints in each case. Of the 30 breakpoints, 16 were located in Alu elements, revealing non-allelic homologous recombination (NAHR) as the putative mechanism for the deletions of the STK11 gene, which lays in a region with high Alu density. In the remaining cases, other mechanisms could be hypothesized, such as microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). In conclusion we here demonstrated the non-random occurrence of large deletions associated with PJS. All our patients had a classical PJS phenotype, which shows that haploinsufficiency for SBNO2, C19orf26, ATP5D, MIDN, C19orf23, CIRBP, C19orf24,and EFNA2, does not apparently affect their clinical phenotype.

An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5' splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the mutated 5' splice site to several cryptic, noncanonical 3' splice sites immediately adjacent to the normal 3' splice site. In vitro splicing analysis demonstrates that this aberrant splicing is mediated by the U12-dependent spliceosome. The results indicate that the minor spliceosome can use a variety of 3' splice site sequences to pair to a given 5' splice site, albeit with tight constraints for maintaining the 3' splice site position. The unusual splicing defect associated with this PJS-causing mutation uncovers differences in splice-site recognition between the major and minor pre-mRNA splicing pathways.

Effects of VEGF on phenotypic severity in children with hereditary hemorrhagic telangiectasia.

The purpose of this study was to estimate vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta1 serum levels in children with hereditary hemorrhagic telangiectasia (HHT) type 1 and type 2 and to correlate them to the presence of arteriovenous malformations (AVMs). High VEGF levels were initially found in an infant who had been hospitalized with intestinal bleeding and suspected HHT. This case led to the evaluation of VEGF and TGF-beta1 by standard enzyme-linked immunosorbent assay in 13 children with HHT and familiarity. Patients were divided into 2 groups on the basis of the presence/absence of pulmonary AVMs. No significant difference was found for VEGF and TGF-beta1 levels in HHT patients versus controls. Among HHT patients, serum levels of VEGF in those without AVM were significantly lower than those with AVM and normal controls. No difference for TGF-beta1 levels was found in these patient subgroups. Low VEGF levels may represent a protection factor against the onset of pulmonary AVMs in HHT children. However, neither VEGF nor TGF-beta1 can be used as biochemical markers for an early diagnosis in HHT. The diagnosis of HHT still requires clinical criteria, which permitted to confirm the presence of the disease in the infant with intestinal bleeding.

SCE frequency measurement could be useful in the prenatal diagnosis of Roberts syndrome.

In a previously published article (Resta et al., 2006) on Robert's syndrome in prenatal diagnosis, a case of a 36-year-old woman and her 36-year-old, nonconsanguineous husband were presented. Our findings suggest the existence of nonsense mediated decay (NMD) variability which could account for the varying severity reported in carriers of identical mutations. Furthermore, fetal cells were used to evaluate the influence of premature centromere separation (PCS) on the sister chromatid exchange (SCE) and micronucleus (MN) frequency. Given the similar variation observed in the SCE frequencies, dependent on tissue/cell type (amniotic fluid sample, chorionic villus sampling) and duration of in vitro cultures (48 hours or 72 hours), the idea was that this new piece of information could be interesting. It seems that the SCE frequency increased proportionally to the cell cycle increasing (1 degrees < 2 degrees < 3 degrees ... n). Obviously, our observations are too scarce to draw conclusions, but further investigation could be useful to corroborate or dispute these results, considering that the two techniques, (MN and SCE), are simple to perform and do not require expensive laboratory equipment.

DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.

Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation.

In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects.

BACKGROUND: Abnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. However, apart from the mutations in the donor and acceptor sites, the effects on splicing of other sequence variations are difficult to predict. Loosely defined exonic and intronic sequences have been shown to affect splicing efficiency by means of silencing and enhancement mechanisms. Thus, nucleotide substitutions in these sequences can induce aberrant splicing. Web-based resources have recently been developed to facilitate the identification of nucleotide changes that could alter splicing. However, computer predictions do not always correlate with in vivo splicing defects. The issue of unclassified variants in cancer predisposing genes is very important both for the correct ascertainment of cancer risk and for the understanding of the basic mechanisms of cancer gene function and regulation. Therefore we aimed to verify how predictions that can be drawn from in silico analysis correlate with results obtained in an in vivo splicing assay. RESULTS: We analysed 99 hMLH1 and hMSH2 missense mutations with six different algorithms. Transfection of three different cell lines with 20 missense mutations, showed that a minority of them lead to defective splicing. Moreover, we observed that some exons and some mutations show cell-specific differences in the frequency of exon inclusion. CONCLUSION: Our results suggest that the available algorithms, while potentially helpful in identifying splicing modulators especially when they are located in weakly defined exons, do not always correspond to an obvious modification of the splicing pattern. Thus caution must be used in assessing the pathogenicity of a missense or silent mutation with prediction programs. The variations observed in the splicing proficiency in three different cell lines suggest that nucleotide changes may dictate alternative splice site selection in a tissue-specific manner contributing to the widely observed phenotypic variability in inherited cancers.

Tumor-specific hyperactive low-molecular-weight cyclin E isoforms detection and characterization in non-metastatic colorectal tumors.

PURPOSE: Several molecules involved in cancer biology have been studied as potential prognostic markers. Recently, overexpression of cyclin E and its low-molecular-weight (LMW) isoforms has been reported to be the most prominent prognostic marker in breast cancer, surpassing proliferation index, ploidy, and axillary nodal involvement. Furthermore, cyclin E and p53 are considered the main factors controlling the euploid equilibrium in human cells. We investigated the status of cyclin E and p53 in cell lines and tissue samples of colorectal cancer, one of the leading causes of death from a tumor in the Western world. EXPERIMENTAL DESIGN: We analyzed colorectal cancer cells, from established cell lines and patient specimens, to determine the protein levels of cyclin E and p53, and to detect p53 and APC mutations, microsatellite and chromosome instability. In addition, we assessed the presence of cyclin E LMW isoforms and their enzymatic activity. RESULTS: Colorectal cancer cells expressed hyperactive LMW forms both in vitro and in vivo. These tumor-specific isoforms are correlated to genomic instability even in p53-proficient cells, and represented a constant feature in the tumors analyzed. CONCLUSIONS: In colorectal cancer, the formation of cyclin E LMW forms is an early event leading to DNA-damage checkpoint-independent proliferation. Collectively, our results provide evidence that evaluation of LMW forms could represent a novel tool in the molecular characterization of colorectal tumors aimed at identifying sensitive prognostic factors and uncovering subsets of high-risk patients within the traditional categories.

Activation of MyoD-dependent transcription by cdk9/cyclin T2.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101-161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

Association of beta-adrenergic receptor polymorphisms and progression to heart failure in patients with idiopathic dilated cardiomyopathy.

PURPOSE: Increased sympathetic nervous system activation via the beta-adrenergic pathway influences the evolution of idiopathic dilated cardiomyopathy. We assessed the effects of beta-adrenergic receptor variants on heart failure in idiopathic dilated cardiomyopathy. METHODS: We prospectively analyzed 171 consecutive patients (mean [+/- SD] age, 49 +/- 14 years; 129 men) with idiopathic dilated cardiomyopathy who were receiving conventional treatment. All were characterized by polymerase chain reaction-restriction fragment length polymorphism analysis for Ser49Gly and Arg389Gly in the beta1-adrenergic receptor; the 5' leader cistron (LC) Arg19Cys, Arg16Gly, Gln27Glu, and Thr164Ile in the beta2-adrenergic receptor; and Arg64Trp in the beta3-adrenergic receptor. The endpoint was heart failure, defined as a worsening of clinical condition leading to hospitalization for heart failure, cardiac transplantation, or death from heart failure. RESULTS: During a median follow-up of 33 months, 24 patients had heart failure. In a Cox univariate analysis, the beta1Gly49 and beta2 5'LC-Cys19, Arg16, and Gln27 alleles were associated with a lower risk of heart failure. In a multivariate analysis that considered age, functional class, left ventricular ejection fraction, and beta-blocker use, three beta2-adrenergic receptor alleles were associated with lower risk: 5'LC-Cys19 (hazard ratio [HR]: 0.15; 95% confidence interval [CI]: 0.05 to 0.42), Arg16 (HR: 0.12; 95% CI: 0.04 to 0.35), and Gln27 (HR: 0.15; 95% CI: 0.05 to 0.42). CONCLUSION: The Gly49 allele in the beta1-adrenergic receptor and the 5' LC-Cys19, Arg16, and Gln27 alleles in the beta2-adrenergic receptor were associated with a lower risk of heart failure in idiopathic dilated cardiomyopathy, suggesting that the beta1- and beta2-adrenergic receptor genes are modifier genes.

The Italian external quality control program for familial adenomatous polyposis of the colon: five years of experience.

Familial adenomatous polyposis is a rare autosomal dominant inherited disease (incidence, 1/8000). More than 90% of families affected by familial adenomatous polyposis have a mutation in the tumor suppressor gene adenomatous polyposis coli (APC). Mutations in this gene are characterized by 100% penetrance, although there is a variation in phenotypic expression of the disease. According to a 2004 survey of the Italian Human Genetic Society, about 264 APC gene molecular genetic tests were performed by Italian laboratories per year. The Italian External Quality Assessment (IEQA), financially supported by the Ministry of Health and coordinated by the Istituto Superiore di Sanità, was started in 2000 to improve the quality of molecular genetic tests in Italy. In the frame of the IEQA, about 50% of public laboratories performing APC gene tests have been monitored. The number of responding public laboratories during the 5 years was 6, 7, 7, 7, and 5 from 2001 to 2006, respectively; on average, 96.3% of samples completely analyzed were correctly genotyped. Methods used by laboratories to detect mutation were direct sequencing, single-strand conformation polymorphism, protein truncation test, and denaturing high-performance liquid chromatography. Written reports were not homogeneous among laboratories, although a new form of written report was proposed to laboratories in 2004. It will be interesting to monitor the effects of the reporting model and the output of this educational action in the future.

Influence of some detoxification enzyme polymorphisms on cytogenetic biomarkers between individuals exposed to very low doses of 1,3-butadiene.

OBJECTIVE: To evaluate the variation of some biomarkers related to the level of enzymatic activity dependent on the different polymorphisms. METHODS: We studied 27 butadiene-exposed workers and 37 controls using different biomarkers of the genotoxic effect. The genotypes were determined using polymerase chain reaction and restriction fragment length polymorphism-polymerase chain reaction techniques; the subjects were assigned to a specific group based on the microsomal epoxide hydrolase (mEH) activity predicted by their genotype (low, intermediate, high). RESULTS: The studied biomarkers were not able to discriminate between exposed and control individuals, but sister chromatid exchange (SCE) and high frequency cells were influenced by smoking habits. Smokers having fast microsomal epoxide hydrolase activity showed higher SCE frequency (7.61) respect to those presenting intermediate (5.86) or slow (6.65) enzymatic activity. CONCLUSIONS: On the basis of these results, can we suppose the existence of an "intermediate genotype" advantage (at least for induction of SCE)?

De novo balanced chromosome rearrangements in prenatal diagnosis.

OBJECTIVE: We surveyed the datasheets of 29 laboratories concerning prenatal diagnosis of de novo apparently balanced chromosome rearrangements to assess the involvement of specific chromosomes, the breakpoints distribution and the impact on the pregnancy outcome. METHOD: By means of a questionnaire, data on 269.371 analyses performed from 1983 to 2006 on amniotic fluid, chorionic villus and fetal blood samples were collected. RESULTS: A total of 246 balanced anomalies were detected at frequencies of 72% for reciprocal translocations, 18% for Robertsonian translocations, 7% for inversions and 3% for complex chromosome rearrangements. The total frequencies of balanced rearrangements were 0.09%, 0.08% and 0.05% on amniotic fluid, chorionic villus and fetal blood samples. CONCLUSION: A preferential involvement of chromosomes 22, 7, 21, 3, 9 and 11 and a less involvement of chromosomes X, 19, 12, 6 and 1 was observed. A nonrandom distribution of the breakpoints across chromosomes was noticed. Association in the location of recurrent breakpoints and fragile sites was observed for chromosomes 11, 7, 10 and 22, while it was not recorded for chromosome 3. The rate of pregnancy termination was about 20%, with frequencies decreasing from complex chromosomal rearrangements (33%), reciprocal translocations (24%) to inversions (11%) and Robertsonian translocations (3%).

Porous silicon surfaces: a candidate substrate for reverse protein arrays in cancer biomarker detection.

This paper introduces a new substrate for reverse-phase protein microarray applications based on macroporous silicon. A key feature of the microarray substrate is the vastly surface enlarging properties of the porous silicon, which simultaneously offers highly confined microarray spots. The proof of principle of the reverse array concept was demonstrated in the detection of different levels of cyclin E, a possible cancer biomarker candidate which regulates G1-S transition and correlates with poor prognosis in different types of human cancers. The substrate properties were studied performing analysis of total cyclin E expression in human colon cancer cell lines Hct116 and SW480. The absence of unspecific binding and good microarray quality was demonstrated. In order to verify the performance of the 3-D textured macroporous surface for complex biological samples, lysates of the human tissue spiked to different levels with cell extract overproducing cyclin E (Hct116) were arrayed on the chip surface. The samples were spotted in a noncontact mode in 100 pL droplets with spots sizes ranged between 50 and 70 mum and spot-to-spot center distances 100 mum, allowing microarray spot densities up to 14 000 spots per cm(2). The different sample types of increasing complexities did not have any impact on the spot intensities recorded and the protein spots showed good homogeneity and reproducibility over the recorded microarrays. The data demonstrate the potential use of macroporous silicon as a substrate for quantitative determination of a cancer biomarker cyclin E in tissue lysates.

Molecular and clinical characteristics in 46 families affected with Peutz-Jeghers syndrome.

Germline mutations of the tumor suppressor gene LKB1/STK11 are responsible for the Peutz-Jeghers syndrome (PJS), an autosomal-dominant disorder characterized by mucocutaneous pigmentation, hamartomatous polyps, and an increased risk of associated malignancies. In this study, we assessed the presence of pathogenic mutations in the LKB1/STK11 gene in 46 unrelated PJS families, and also carried genotype-phenotype correlation in regard of the development of cancer in 170 PJS patients belonging to these families. All LKB1/STK11 variants detected with single-strand conformational polymorphism were confirmed by direct sequencing, and those without LKB1/STK11 mutation were further submitted to Southern blot analysis for detection of deletions/rearrangements. Statistical analysis for genotype-phenotype correlation was performed. In 59% (27/46) of unrelated PJS cases, pathogenic mutations in the LKB1/STK11 gene, including 9 novel mutations, were identified. The new mutations were 2 splice site deletion-insertions, 2 missenses, 1 nonsense, and 4 abnormal splice sites. Genotype-phenotype analysis did not yield any significant differences between patients carrying mutations in LKB1/STK11 versus those without mutations, even with respect to primary biliary adenocarcinoma. This study presents the molecular characterization and cancer occurrence of a large cohort of PJS patients, increases the mutational spectrum of LKB1/STK11 allelic variants worldwide, and provides a new insight useful for clinical diagnosis and genetic counseling of PJS families.

Structural variation on the short arm of the human Y chromosome: recurrent multigene deletions encompassing Amelogenin Y.

Structural polymorphism is increasingly recognized as a major form of human genome variation, and is particularly prevalent on the Y chromosome. Assay of the Amelogenin Y gene (AMELY) on Yp is widely used in DNA-based sex testing, and sometimes reveals males who have interstitial deletions. In a collection of 45 deletion males from 12 populations, we used a combination of sequence-tagged site mapping, and binary-marker and Y-short tandem repeat haplotyping to understand the structural basis of this variation. Of the 45 deletion males, 41 carry indistinguishable deletions, 3.0-3.8 Mb in size. Breakpoint mapping strongly implicates a mechanism of non-allelic homologous recombination between the proximal major array of TSPY gene-containing repeats, and a single distal copy of TSPY; this is supported by the estimation of TSPY copy number in deleted and non-deleted males. The remaining four males carry three distinct non-recurrent deletions (2.5-4.0 Mb), which may be due to non-homologous mechanisms. Haplotyping shows that TSPY-mediated deletions have arisen seven times independently in the sample. One instance, represented by 30 chromosomes mostly of Indian origin within haplogroup J2e1*/M241, has a time-to-most-recent-common-ancestor of approximately 7700+/-1300 years. In addition to AMELY, deletion males all lack the genes PRKY and TBL1Y, and the rarer deletion classes also lack PCDH11Y. The persistence and expansion of deletion lineages, together with direct phenotypic evidence, suggests that absence of these genes has no major deleterious effects.

Pulmonary arteriovenous malformations, hereditary hemorrhagic telangiectasia, and brain abscess.

Hereditary hemorrhagic telangiectasia (HHT) is a systemic angiodysplasia inherited as an autosomal dominant disease. Patients with HHT and pulmonary arteriovenous malformations (PAVMs) are at increased risk for brain abscess (BA), a potentially preventable condition as effective treatment for PAVMs is available. In a center dedicated to HHT, a history of BA was found in 6 out of 128 patients with a definite diagnosis: herewith, their histories are reported focusing on mistakes in the diagnosis and management of the disease. Patients with PAVMs and BA had a higher mean hemoglobin concentration (15.1 g/dl vs. 12.2 g/dl, p < 0.006 by Student's t test) compared to patients with PAVMs alone. Other clinical features (genetics, bacteriology, types of PAVMs, treatments, outcomes) are also discussed. Prompt diagnosis and screening for visceral involvement is pivotal for HHT patients and their relatives.

Lack of genotoxic effect in workers exposed to very low doses of 1,3-butadiene.

1,3-Butadiene (BD), a probable carcinogen to humans, has been shown to have an ill-defined genotoxicity in occupationally exposed workers. In the present study, the influence of exposure to very low doses of BD and to cigarette smoking was investigated on some cytogenetic endpoints, namely, sister chromatid exchanges (SCE), chromosomal aberrations (CA) and cells with a high frequency of SCE (HFC), in peripheral blood lymphocytes. Twenty-seven male workers employed in a petrochemical plant and 26 matched controls were included in the study. As regards the airborne BD values, there was a significant difference between exposed (median BD value 1.5, min-max 0.2-69.0 microg/m3) and non-exposed workers (median BD value 0.4, min-max <0.1-3.8 microg/m3). Genotoxic biomarkers were not able to distinguish between the two groups. The frequency of SCE was higher in smokers than in non-smokers (p=0.001), with a positive correlation between the number of cigarettes smoked per day and both SCE (r=0.4; p=0.004) and HFC frequency (r=0.3; p=0.04). Multiple regression analysis confirmed the influence of cigarette smoking on the level of SCE and HFC, while these parameters were not affected by personal exposure to BD. Overall, the biomarkers of genotoxic effect investigated in our study were not able to discriminate between workers with a very low exposure to BD and controls, while it was possible to distinguish between smokers and non-smokers on the basis of SCE.