Nicoletta Resta

Analysis of the LKB1-STRAD-MO25 complex

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25.

Cancer risks in LKB1 germline mutation carriers

Background and aims: Germline mutations in the LKB1 gene are known to cause Peutz-Jeghers syndrome, which is an autosomal dominant disorder characterised by hamartomatous polyposis and mucocutaneous pigmentation. This syndrome is associated with an increased risk of malignancies in different organs but there is a lack of data on cancer range and risk in LKB1 germline mutation carriers. Patients and methods: The cumulative incidence of cancer in 149 Peutz-Jeghers syndrome patients with germline mutation(s) in LKB1 was estimated using Kaplan-Meier time to cancer onset analyses and compared between relevant subgroups with log rank tests. Results: Thirty two cancers were found in LKB1 mutation carriers. Overall cancer risks at ages 30, 40, 50, 60, and 70 years were 6%, 18%, 31%, 41%, and 67%, respectively. There were similar overall cancer risks between male and female carriers. However, there were overall cancer risk differences for exon 6 mutation carriers versus non-exon 6 mutation carriers (log rank p = 0.022 overall, 0.56 in males, 0.0000084 in females). Most (22/32) of the cancers occurred in the gastrointestinal tract, and the overall gastrointestinal cancer risks at ages 40, 50, 60, and 70 years were 12%, 24%, 34%, and 63%, respectively. In females, the risks for developing gynaecologic cancer at ages 40 and 50 years were 13% and 18%, respectively. Conclusions: Mutations in exon 6 of LKB1 are associated with a higher cancer risk than mutations within other regions of the gene. Moreover, this study provides age related cumulative risks of developing cancer in LKB1 mutation carriers that should be useful for developing a tailor made cancer surveillance protocol for Peutz-Jeghers syndrome patients.

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.

A homozygous frameshift mutation in the ESCO2 gene: Evidence of intertissue and interindividual variation in Nmd efficiency

Roberts syndrome (RS) is a rare disorder characterized by tetraphocomelia and several other clinical features. Cells from RS patients exhibit characteristic premature separation of heterochromatic region of many chromosomes and abnormalities in cell cycle. Mutations in the ESCO2 gene have recently been identified in 20 RS families. We performed mutational analysis of the ESCO2 gene in two fetuses diagnosed with RS and their normal parents. In both fetuses, we identified homozygosity for the c. 745_746delGT mutation, while the non‐consanguineous parents were both heterozygous for the same mutation. Considering the position of the mutation identified, we carried out qualitative and quantitative real‐time ESCO2 cDNA analysis on RNA isolated from CVS‐stromal cells in one fetus, amniocytes in the second fetus, and lymphocytes from the heterozygous parents. The results of this analysis showed that despite the presence of a premature termination codon (PTC) 112 nucleotides upstream of the next exon3–exon4 junction, the mutant ESCO2 mRNA was present in both fetuses, albeit at low levels, indicating a partial resistance to nonsense mediated decay (NMD). Interestingly, when cells derived from the two fetuses were treated with an inhibitor of translation, they revealed the presence of tissue and individual variability in NMD efficiency, despite the identical mutational status. The existence of such a variation in the NMD efficiency could explain the broad intrafamilial and interfamilial variability in the clinical presentation of RS patients, and in other genetic diseases where nonsense mutations are responsible for most of the mutation load. Moreover, considering that a mutated full length mRNA was produced in both fetuses, we used Western blot analysis to demonstrate the absence of the ESCO2‐truncated protein in cells derived from both fetuses and in a lymphoblastoid cell line derived from the parents. J. Cell. Physiol. 209: 67–73, 2006.

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.

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.

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.

Phosphatase and Tensin Homolog (PTEN) Gene Mutations and Autism: Literature Review and a Case Report of a Patient With Cowden Syndrome, Autistic Disorder, and Epilepsy

Phosphatase and tensin homolog (PTEN) gene mutations are associated with a spectrum of clinical disorders characterized by skin lesions, macrocephaly, hamartomatous overgrowth of tissues, and an increased risk of cancers. Autism has rarely been described in association with these variable clinical features. At present, 24 patients with phosphatase and tensin homolog gene mutation, autism, macrocephaly, and some clinical findings described in phosphatase and tensin homolog syndromes have been reported in the literature. We describe a 14-year-old boy with autistic disorder, focal epilepsy, severe and progressive macrocephaly, and multiple papular skin lesions and palmoplantar punctate keratoses, characteristic of Cowden syndrome. The boy has a de novo phosphatase and tensin homolog gene mutation. Our patient is the first case described to present a typical Cowden syndrome and autism associated with epilepsy.

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.

COQ4 Mutations Cause a Broad Spectrum of Mitochondrial Disorders Associated with CoQ10 Deficiency

Primary coenzyme Q10 (CoQ10) deficiencies are rare, clinically heterogeneous disorders caused by mutations in several genes encoding proteins involved in CoQ10 biosynthesis. CoQ10 is an essential component of the electron transport chain (ETC), where it shuttles electrons from complex I or II to complex III. By whole-exome sequencing, we identified five individuals carrying biallelic mutations in COQ4. The precise function of human COQ4 is not known, but it seems to play a structural role in stabilizing a multiheteromeric complex that contains most of the CoQ10 biosynthetic enzymes. The clinical phenotypes of the five subjects varied widely, but four had a prenatal or perinatal onset with early fatal outcome. Two unrelated individuals presented with severe hypotonia, bradycardia, respiratory insufficiency, and heart failure; two sisters showed antenatal cerebellar hypoplasia, neonatal respiratory-distress syndrome, and epileptic encephalopathy. The fifth subject had an early-onset but slowly progressive clinical course dominated by neurological deterioration with hardly any involvement of other organs. All available specimens from affected subjects showed reduced amounts of CoQ10 and often displayed a decrease in CoQ10-dependent ETC complex activities. The pathogenic role of all identified mutations was experimentally validated in a recombinant yeast model; oxidative growth, strongly impaired in strains lacking COQ4, was corrected by expression of human wild-type COQ4 cDNA but failed to be corrected by expression of COQ4 cDNAs with any of the mutations identified in affected subjects. COQ4 mutations are responsible for early-onset mitochondrial diseases with heterogeneous clinical presentations and associated with CoQ10 deficiency.