A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover

peer-reviewedBackground: White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. Results: We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. Conclusions: In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a “core set” of ancestrally derived homeolog specific variants in white clover.Department of Agriculture Food & the Marine, Ireland - Research Stimulus Fund (RSF 07–566

LKB1/KRAS mutant lung cancers constitute a genetic subset of NSCLC with increased sensitivity to MAPK and mTOR signalling inhibition

LKB1/STK11 is a multitasking tumour suppressor kinase. Germline inactivating mutations of the gene are responsible for the Peutz-Jeghers hereditary cancer syndrome. It is also somatically inactivated in approximately 30% of non-small-cell lung cancer (NSCLC). Here, we report that LKB1/KRAS mutant NSCLC cell lines are sensitive to the MEK inhibitor CI-1040 shown by a dose-dependent reduction in proliferation rate, whereas LKB1 and KRAS mutations alone do not confer similar sensitivity. We show that this subset of NSCLC is also sensitised to the mTOR inhibitor rapamycin. Importantly, the data suggest that LKB1/KRAS mutant NSCLCs are a genetically and functionally distinct subset and further suggest that this subset of lung cancers might afford an opportunity for exploitation of anti-MAPK/mTOR-targeted therapies

Mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADα, and MO25α, in Peutz–Jeghers syndrome

Mutations in LKB1 lead to Peutz–Jeghers syndrome (PJS). However, only a subset of PJS patients harbours LKB1 mutations. We performed a mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADα, and MO25α, in 28 LKB1-negative PJS patients. No disease-causing mutations were detected in the studied genes in PJS patients from different European populations

Mutations in STK11 gene in Czech Peutz-Jeghers patients

<p>Abstract</p> <p>Background</p> <p>Peutz-Jeghers syndrome (PJS) is an autosomal dominant hereditary disease characterized by mucocutaneous pigmentation and gastrointestinal hamartomatous polyposis. The germline mutations in the serine/threonine kinase 11 (<it>STK11</it>) gene have been shown to be associated with the disease. Individuals with PJS are at increased risk for development of various neoplasms. The aim of the present study was to characterize the genotype and phenotype of Czech patients with PJS.</p> <p>Methods</p> <p>We examined genomic DNA of 8 individuals from five Czech families by sequencing analysis of <it>STK11 </it>gene, covering its promotor region, the entire coding region and the splice-site boundaries, and by multiplex ligation-dependent probe amplification (MLPA) assay designed for the identification of large exonic deletions or duplications of <it>STK11 </it>gene.</p> <p>Results</p> <p>We found pathogenic mutations in <it>STK11 </it>gene in two families fulfilling the diagnostic criteria of PJS and in one of three sporadic cases not complying with the criteria. The patient with the frameshift mutation in <it>STK11 </it>gene developed aggressive gastric cancer. No other studied proband has developed a carcinoma so far.</p> <p>Conclusion</p> <p>Our results showed that a germline mutation of <it>STK11 </it>gene can be found not only in probands fulfilling the PJS diagnostic criteria, but also in some sporadic cases not complying with the criteria. Moreover, we observed a new case of aggressive gastric cancer in a young patient with a frameshift mutation of <it>STK11 </it>gene.</p

PTEN status is a crucial determinant of the functional outcome of combined MEK and mTOR inhibition in cancer

Combined MAPK/PI3K pathway inhibition represents an attractive, albeit toxic, therapeutic strategy in oncology. Since PTEN lies at the intersection of these two pathways, we investigated whether PTEN status determines the functional response to combined pathway inhibition. PTEN (gene, mRNA, and protein) status was extensively characterized in a panel of cancer cell lines and combined MEK/mTOR inhibition displayed highly synergistic pharmacologic interactions almost exclusively in PTEN-loss models. Genetic manipulation of PTEN status confirmed a mechanistic role for PTEN in determining the functional outcome of combined pathway blockade. Proteomic analysis showed greater phosphoproteomic profile modification(s) in response to combined MEK/mTOR inhibition in PTEN- loss contexts and identified JAK1/STAT3 activation as a potential mediator of synergistic interactions. Overall, our results show that PTEN-loss is a crucial determinant of synergistic interactions between MAPK and PI3K pathway inhibitors, potentially exploitable for the selection of cancer patients at the highest chance of benefit from combined therapeutic strategies

Gene for the human transmembrane-type protein tyrosine phosphatase H (PTPRH): genomic structure, fine-mapping and its exclusion as a candidate for Peutz-Jeghers syndrome

Mutations in the serine/threonine kinase STK11 lead to Peutz-Jeghers syndrome (PJS) in a subset of affected individuals. Significant evidence for linkage to a second potential PJS disease locus on 19q13.4 has previously been described in one PJS family (PJS07). In the current study, we investigated this second locus for PJS gene candidates. We mapped the main candidate gene in this region, the gene for the transmembrane-type protein tyrosine phosphatase H (PTPRH), within 15 kb telomeric to the marker D19S880. We determined its genomic structure, and performed mutation analysis of all exons and the exon-intron junctions of the PTPRH gene in the PJS07 family. No disease causing mutation was identified in PTPRH in affected individuals, suggesting the existence of an as yet not identified gene on 19q13.4 as a second PJS gene

The gene for autosomal dominant hidrotic ectodermal dysplasia (Clouston syndrome) in a large Indian family maps to the 13q11-q12.1 pericentromeric region

Hidrotic ectodermal dysplasia (HED), Clouston syndrome (MIM No. 129500), is an autosomal dominant disorder affecting the skin and its derivatives. It is characterized by alopecia, dysplastic nails in hands and feet, and hyperkeratosis of the palms and soles. We have studied a large Indian pedigree (UR005), from Gujarat region, consisting of a total 127 individuals including 41 affected (12 males and 29 females). The phenotype in this family ranged from atrichosis to hypotrichosis, sparsity or absence of eyebrows, and thickening of palms and soles. In order to map the disease locus by linkage analysis, DNA polymorphisms were used in DNAs from 23 affected and 8 normal individuals. While genotyping was in progress, Kibar et al. [1996] reported mapping of the locus of a similar disease in French-Canadian families to 13q around marker D13S141. We then utilized markers on 13q to genotype the members of the Indian family. Linkage with 13q11-12.1 markers was confirmed with a maximum lod score of 3.27 (theta=0.00) with locus D13S1316. Multipoint linkage analysis yielded a lod score of 5.04 at theta=0.00 with D13S1316; haplotype analysis indicated that the gene for the Clouston syndrome in this family is localized proximal to D13S292. These data suggest that the gene for the Clouston syndrome in this Indian pedigree is probably the same as that described in the French Canadian families. The combination of data from all available families linked to 13q11-12.1 will make it possible to narrow the critical region and facilitate the positional cloning of the elusive gene