Patrizia Lastella

Hereditary hemorrhagic telangiectasia: clinical features in ENG and ALK1 mutation carriers

Summary.  Background: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by epistaxis, mucocutaneous telangiectases and visceral arteriovenous malformations (AVMs), particularly in the brain (CAVMs), lungs (PAVMs), liver (HAVMs) and gastrointestinal tract (GI). The identification of a mutated ENG (HHT1) or ALK‐1 (HHT2) gene now enables a genotype–phenotype correlation. Objective: To determine the incidence of visceral localizations and evaluate phenotypic differences between ENG and ALK1 mutation carriers. Methods: A total of 135 consecutive adult patients were subjected to mutational screening in ENG and ALK1 genes and instrumental tests to detect AVMs, such as chest–abdomen multislice computed tomography (MDCT), brain magnetic resonance imaging and magnetic resonance angiography (MRI/MRA), upper endoscopy, were offered to all patients, independent of presence of clinical symptoms. The 122 patients with identified mutations were enrolled in the study and genotype–phenotype correlations were established. Results: PAVMs and CAVMs were significantly more frequent in HHT1 (75% vs. 44%, P < 0.0005; 20% vs. 0%, P < 0.002, respectively) and HAVMs in HHT2 (60% vs. 84%, P < 0.01). No age difference was found for PAVMs whereas HAVMs were significantly higher in older patients in both HHT1 and HHT2. Neurological manifestations secondary to CAVMs/PAVMs were found only in HHT1 patients, whereas severe liver involvement was detected only in HHT2. Respiratory symptoms were mainly detected in HHT1. Conclusions: Our study evidences a higher visceral involvement in HHT1 and HHT2 compared with previous reports. HHT1 is more frequently associated with congenital AVM malformations, such as CAVMs and PAVMs whereas HHT2 predominantly involves the liver. The ENG gene should be first targeted for mutational screening in the presence of large PAVM in patients < 45 years.

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

BackgroundAbnormalities 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.ResultsWe 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.ConclusionOur 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.

Cancer risk associated with STK11/LKB1 germline mutations in Peutz-Jeghers syndrome patients: Results of an Italian multicenter study

BACKGROUND Germline mutations in the STK11/LKB1 gene cause Peutz-Jeghers syndrome, an autosomal-dominantly inherited condition characterized by mucocutaneous pigmentation, hamartomatous gastrointestinal polyposis, and an increased risk for various malignancies. We here report the results of the first Italian collaborative study on Peutz-Jeghers syndrome. AIMS To assess cancer risks in a large homogenous cohort of patients with Peutz-Jeghers syndrome, carrying, in large majority, an identified STK11/LKB1 mutation. METHODS One-hundred and nineteen patients with Peutz-Jeghers syndrome, ascertained in sixteen different Italian centres, were enrolled in a retrospective cohort study. Relative and cumulative cancer risks and genotype-phenotype correlations were evaluated. RESULTS 36 malignant tumours were found in 31/119 (29 STK11/LKB1 mutation carriers) patients. The mean age at first cancer diagnosis was 41 years. The relative overall cancer risk was 15.1 with a significantly higher risk (p < 0.001) in females (22.0) than in males (8.6). Highly increased relative risks were present for gastrointestinal (126.2) and gynaecological cancers (27.7), in particular for pancreatic (139.7) and cervical cancer (55.6). The Kaplan-Meier estimates for overall cumulative cancer risks were 20%, 43%, 71%, and 89%, at age 40, 50, 60 and 65 years, respectively. CONCLUSION Peutz-Jeghers syndrome entails markedly elevated cancer risks, mainly for pancreatic and cervical cancers. This study provides a helpful reference for improving current surveillance protocols.

Germline novel MSH2 deletions and a founder MSH2 deletion associated with anticipation effects in HNPCC

Hereditary non‐polyposis colorectal cancer (HNPCC) is caused by inactivating mutations of DNA mismatch repair genes. Large genomic rearrangements in these genes have been increasingly recognized as important causes of HNPCC. Using multiplex ligation‐dependent probe amplification, we identified three MSH2 deletions in Italian patients with HNPCC (proband A: exons 1–3, proband M: exon 8, and proband C: exons 1–6). Deletion breakpoint sequencing allowed us to develop rapid polymerase chain reaction‐based mutation screening, which confirmed the presence of the deletions in affected and asymptomatic individuals of families A, C, and M. While the exon 8 and exon 1–3 deletions appear to be novel, the MSH2 1–6 deletion found in family C is identical to the one recently documented in two branches of another unrelated Italian family (family V+Va). Haplotype analysis showed that the kindreds C and V+Va (both from northeastern Italy, both displaying clinical features of the Muir–Torre syndrome) shared a seven‐locus haplotype, indicating that the MSH2 1–6 deletion is probably a founder mutation. Families A, C, M, and V+Va all showed progressively earlier cancer‐onset ages in successive generations. Analysis of 23 affected parent–child pairs in the four kindreds showed median anticipation of 12 years in offsprings’ onset of cancer (p = 0.0001). No birth cohort effect was found. This is the first significant evidence of anticipation effects in HNPCC families carrying MSH2 deletions.

Screening for children from families with Rendu–Osler–Weber disease: from geneticist to clinician

Summary.  Background: Rendu–Osler–Weber syndrome, or hereditary hemorrhagic telangiectasia (HHT), is an autosomal dominant vascular disorder. The syndrome is characterized by telangiectases and arteriovenous malformations (AVMs) affecting skin, mucosae and internal organs. AVMs often remain clinically silent until provoking sudden serious complications, responsible for important morbidity and mortality which can occur both in adulthood and in children. The incidence of AVMs in HHT pediatric populations is unknown. Objective: To describe the screening protocol performed in the first genotypically confirmed HHT pediatric population and to estimate the incidence of occult brain, lung and liver AVMs and the different disease phenotypes. Materials and methods: Molecular analysis was performed on 35 children, both symptomatic and asymptomatic, who were family members of probands with a previously identified mutation. Clinical‐instrumental examination was performed on the mutation positive cases. Nasal telangiectases were investigated by anterior rhinoscopy. Contrast echocardiography and high resolution thoracic multislice computed tomography (CT) were performed to detect pulmonary arteriovenous malformations (PAVMs), and echo‐color Doppler, and abdominal CT to detect hepatic arteriovenous malformations (HAVMs). Brain magnetic resonance imaging was utilized to detect cerebral angiopathic involvement. Results: Molecular analysis demonstrated the mutation‐carrier status in 22/35 children. Nineteen children, 12 of whom had epistaxis, positive to molecular testing underwent clinical evaluation. Nasal teleangiectases were found in 68%, mucocutaneous telangiectases (fingers, lips and oral cavity) in 79%, PAVMs in 53%, HAVMs in 47% and cerebral anteriovenous malformations and/or cerebral ischemic changes secondary to PAVMs in 12%. Conclusions: We evidenced a high incidence of HHT children with occult visceral lesions suggesting that a diagnostic screening may be indicated to appropriately treat brain and lung malformations.

Hereditary hemorrhagic telangiectasia: arteriovenous malformations in children.

OBJECTIVE To evaluate the clinical features in a large cohort of pediatric patients with genetically confirmed hereditary hemorrhagic telangiectasia (HHT) and to identify possible predictors of arteriovenous malformation (AVM) onset or clinical significance. STUDY DESIGN Prospective cross-sectional survey of all children subjected to screening for AVMs in the multidisciplinary HHT center. All patients proved to be carriers of endoglin mutations or activin A receptor type-II-like kinase 1 mutations, defined as HHT1 and HHT2, respectively. A full clinical-radiological protocol for AVM detection was adopted, independent from presence or absence of AVM-related symptoms. RESULTS Forty-four children (mean age, 10.3 years; range, 1-18) were subjected to a comprehensive clinical-radiologic evaluation. This investigation disclosed cerebrovascular malformations in 7 of 44 cases, pulmonary AVMs in 20 of 44 cases, and liver AVMs in 23 of 44 cases. Large visceral AVMs were found in 12 of 44 children and were significantly more frequent in patients with HHT1. Only large AVMs were associated with symptoms and complications. CONCLUSIONS Children with HHT have a high prevalence of AVMs; therefore, an appropriate clinical and radiological screening protocol is advisable. Large AVMs can be associated with complications in childhood, whereas small AVMs probably have no clinical risk.

A long diagnostic delay in patients with Hereditary Haemorrhagic Telangiectasia: a questionnaire-based retrospective study

BackgroundThe difficulty in establishing a timely correct diagnosis is a relevant matter of concern for several rare diseases. Many rare-disease-affected patients suffer from considerable diagnostic delay, mainly due to their poor knowledge among healthcare professionals, insufficient disease awareness among patients’ families, and lack of promptly available diagnostic tools. Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomal-dominantly inherited vascular dysplasia, affecting 1:5,000-10,000 patients. HHT is characterized by high variability of clinical manifestations, which show remarkable overlapping with several common diseases.AimTo perform a detailed analysis concerning the diagnostic time lag occurring in patients with HHT, defined as the time period spanning from the first clinical manifestation to the attainment of a definite, correct diagnosis.MethodsA questionnaire was administered to the HHT patients previously recruited from 2000 and 2009. Clinical onset, first referral to a physician for disease manifestations, and first correct diagnosis of definite HHT were collected. Eventual misdiagnosis at first referral and serious complications occurring throughout the time elapsing between disease onset and definite diagnosis were also addressed.ResultsIn the 233 respondents, the clinical onset of disease occurred at an age of 14.1 yrs, while the age of first referral and the age of first definite diagnosis of HHT were 29.2 yrs and 40.1 yrs, respectively. Only 88/233 patients received a correct diagnosis at first counseling. Thus, the diagnostic time lag, represented by the time elapsing from disease onset and first definite diagnosis of HHT, proved to be 25.7 yrs. Twenty-two patients suffered from severe complications during this time interval. The diagnostic delay was significantly longer (p < 0.001) in index patients (first patients who attained definite HHT diagnosis in a given family) than in non-index patients (relative of index patients). The diagnostic time lag was also significantly associated with education grade (p < 0.001).ConclusionsOur data report for the first time a systematic inquiry of diagnostic delay in HHT showing that patients receive a definite diagnosis only after nearly three decades from disease onset. Concerted efforts are still to be made to increase awareness of this disease among both families and physicians.

Molecular and Functional Characterization of Three Different Postzygotic Mutations in PIK3CA-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors.

Background PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed. Methods We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro. Results and Conclusion Our data indicate that patients’ cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.

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.

Endoglin gene mutations and polymorphisms in Italian patients with hereditary haemorrhagic telangiectasia

Autosomal‐dominant hereditary haemorrhagic telangiectasia (HHT) is a genetically heterogeneous disease caused by mutations in at least two different loci. We screened for mutations in four Italian families where segregation studies showed clear evidence of linkage to the endoglin (ENG) locus. In addition, one sporadic case and three patients with pulmonary arteriovenous malformations, belonging to small nuclear families unsuitable for linkage analysis, were included in the screening. The proband from each family was investigated using single‐strand conformation polymorphism and heteroduplex analysis; potential variants were sequenced. Four novel and one previously reported mutation were detected, as well as three new polymorphisms. The novel mutations included deletions in exon 1 (patient 581/02), exon 5 (patient 780/01) and exon 7 (patient 700/01), and a C→T229 substitution in exon 3 (patient 462/02). When analysing patient 700/01 and his affected daughter, we encountered a mutant ENG allele with two mutations – a deletion in exon 7 and a substitution in exon 12 – which converts isoleucine 575 into threonine, in a non‐conserved region. Both mutations were absent in the two healthy sons of the patient, while the polymorphic variant in exon 12 was present in his healthy father. These results and haplotype‐segregation studies suggest that a de novo deletion had occurred in the gamete of paternal origin. For the first time the parental germline in which a de novo HHT mutation occurred has been identified.