A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.

Genetic diagnosis in Lafora disease: genotype-phenotype correlations and diagnostic pitfalls.

Lafora disease (LD) can be diagnosed by skin biopsy, but this approach has both false negatives and false positives. Biopsies of other organs can also be diagnostic but are more invasive. Genetic diagnosis is also possible but can be inconclusive, for example, in patients with only one heterozygous EPM2A mutation and patients with apparently homozygous EPM2B mutations where one parent is not a carrier of the mutation. We sought to identify occult mutations and clarify the genotypes and confirm the diagnosis of LD in patients with apparent nonrecessive disease inheritance. We used single nucleotide polymorphism, quantitative PCR, and fluorescent in situ hybridization analyses. We identified large EPM2A and EPM2B deletions undetectable by PCR in the heterozygous state and describe simple methods for their routine detection. We report a coding sequence change in several patients and describe why the pathogenic role of this change remains unclear. We confirm that adult-onset LD is due to EPM2B mutations. Finally, we report major intrafamilial heterogeneity in age at onset in LD.

Laforin is a cell membrane and endoplasmic reticulum-associated protein tyrosine phosphatase.

Lafora disease (LD) is the only progressive myoclonus epilepsy with polyglucosan bodies. Among conditions with polyglucosan bodies, LD is unique for the subcellular location of its polyglucosans in neuronal perikarya and dendrites and not in axons. Here we report that the protein encoded by the EPM2A gene, which is mutated in LD, localizes at the plasma membrane and the endoplasmic reticulum and that it is a functional protein tyrosine phosphatase. The significance of these findings in the epilepsy of LD and in the origin and characteristic subcellular location of Lafora bodies is discussed.

Mutation spectrum and predicted function of laforin in Lafora's progressive myoclonus epilepsy.

BACKGROUND: Lafora's disease is a progressive myoclonus epilepsy with pathognomonic inclusions (polyglucosan bodies) caused by mutations in the EPM2A gene. EPM2A codes for laforin, a protein with unknown function. Mutations have been reported in the last three of the gene's exons. To date, the first exon has not been determined conclusively. It has been predicted based on genomic DNA sequence analysis including comparison with the mouse homologue. OBJECTIVES: 1) To detect new mutations in exon 1 and establish the role of this exon in Lafora's disease. 2) To generate hypotheses about the biological function of laforin based on bioinformatic analyses. METHODS: 1) PCR conditions and components were refined to allow amplification and sequencing of the first exon of EPM2A. 2) Extensive bioinformatic analyses of the primary structure of laforin were completed. RESULTS: 1) Seven new mutations were identified in the putative exon 1. 2) Laforin is predicted not to localize to the cell membrane or any of the organelles. It contains all components of the catalytic active site of the family of dual-specificity phosphatases. It contains a sequence predicted to encode a carbohydrate binding domain (coded by exon 1) and two putative glucohydrolase catalytic sites. CONCLUSIONS: The identification of mutations in exon 1 of EPM2A establishes its role in the pathogenesis of Lafora's disease. The presence of potential carbohydrate binding and cleaving domains suggest a role for laforin in the prevention of accumulation of polyglucosans in healthy neurons.

Identification of new and common mutations in the EPM2A gene in Lafora disease.

Lafora disease is a teenage onset progressive myoclonus epilepsy caused by mutations in the EPM2A gene. In this report, we describe new mutations within EPM2A, review the known mutations to date to identify the most common, and describe three simple tests for prenatal and carrier screening.

Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism.

Somatic mosaicism due to reversion of a pathogenic allele to wild type has been described in several autosomal recessive disorders. The best known mechanism involves intragenic mitotic recombination or gene conversion in compound heterozygous patients, whereby one allele serves to restore the wild-type sequence in the other. Here we document for the first time functional correction of a pathogenic microdeletion, microinsertion and missense mutation in homozygous Fanconi anaemia (FA) patients resulting from compensatory secondary sequence alterations in cis. The frameshift mutation 1615delG in FANCA was compensated by two additional single base-pair deletions (1637delA and 1641delT); another FANCA frameshift mutation, 3559insG, was compensated by 3580insCGCTG; and a missense mutation in FANCC(1749T-->G, Leu496Arg) was altered by 1748C-->T, creating a cysteine codon. Although in all three cases the predicted proteins were different from wild type, their cDNAs complemented the characteristic hypersensitivity of FA cells to crosslinking agents, thus establishing a functional correction to wild type.

The PISSLRE gene: structure, exon skipping, and exclusion as tumor suppressor in breast cancer.

In sporadic breast cancer, loss of heterozygosity (LOH) frequently occurs in three discrete regions of the long arm of chromosome 16q, the most telomeric of which is located at 16q24.3. Among the genes mapped to this region, PISSLRE is a plausible candidate tumor suppressor gene. It codes for a putative cyclin-dependent kinase that, as with other members of this family, is likely to be involved in regulating the cell cycle and therefore may have a role in oncogenesis. We characterized the genomic structure of PISSLRE and found that the splicing of this gene is complex. A variety of different transcripts were identified, including those due to cryptic splice sites, exon skipping, insertion of intronic sequences, and exon scrambling. The last phenomenon was observed in a rare PISSLRE transcript in which exons are joined at a nonconsensus splice site in an order different from that predicted by the genomic sequence. To screen the PISSLRE gene in breast tumors with ascertained LOH at 16q24.3, we have analyzed each exon by single-strand conformational polymorphism. No variation was found in the coding sequence, leading us to conclude that another tumor suppressor must be targeted by LOH in sporadic breast cancer.

Fine exon-intron structure of the Fanconi anemia group A (FAA) gene and characterization of two genomic deletions.

Fanconi anemia (FA) is a genetically heterogeneous disease with at least eight genes on the basis of complementation groups (FAA to FAH). The analysis of the FAA gene in patients suggested the existence of deletions, none of which have thus far been characterized at the genomic level. A detailed restriction map of the FAA gene with the fine localization of its 43 exons is reported in this paper. We also describe the first two genomic deletions, one of 5.0 kb and another of at least 120 kb. The former was likely the result of a recombination between related Alu sequences. Since these interspersed repeats could generate deletions and insertions by mispairing, rearrangements of this gene are a possibility in those FA families in which FAA mutations have not been identified.

Mutations of the Fanconi anemia group A gene (FAA) in Italian patients.

Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. At least five complementation groups (FA-A-FA-E) have been identified. The relative prevalence of FA-A has been estimated at an average of approximately 65% but may widely vary according to ethnic background. In Italy, 11 of 12 patients analyzed by cell-fusion studies were assigned to group FA-A, suggesting an unusually high relative prevalence of this FA subtype in patients of Italian ancestry. We have screened the 43 exons of the FAA gene and their flanking intronic sequences in 38 Italian FA patients, using RNA-SSCP. Ten different mutations were detected: three nonsense and one missense substitutions, four putative splice mutations, an insertion, and a duplication. Most of the mutations are expected to cause a premature termination of the FAA protein at various sites throughout the molecule. Four protein variants were also found, three of which were polymorphisms. The missense mutation D1359Y, not found in chromosomes from healthy unrelated individuals, was responsible for a local alteration of hydrophobicity in the FAA protein, and it was likely to be pathogenic. Thus, the mutations so far encountered in the FAA gene are essentially all different. Since screening based on the analysis of single exons by genomic DNA amplification apparently detects only a minority of the mutations, methods designed to detect alterations in the genomic structure of the gene or in the FAA polypeptide may be helpful in the identification of FAA mutations.

The genomic organization of the Fanconi anemia group A (FAA) gene.

Fanconi anemia (FA) is a genetically heterogenous disease involving at least five genes on the basis of complementation analysis (FAA to FAE). The FAA gene has been recently isolated by two independent approaches, positional and functional cloning. In the present study we describe the genomic structure of the FAA gene. The gene contains 43 exons spanning approximately 80 kb as determined by the alignment of four cosmids and the fine localization of the first and the last exons in restriction fragments of these clones. Exons range from 34 to 188 bp. All but three of the splice sites were consistent with the ag-gt rule. We also describe three alternative splicing events in cDNA clones that result in the loss of exon 37, a 23-bp deletion at the 5' end of exon 41, and a GCAG insertion at the 3' portion also in exon 41. Sequence analysis of the 5' region upstream of the putative transcription start site showed no obvious TATA and CAAT boxes, but did show a GC-rich region, typical of housekeeping genes. Knowledge of the structure of the FAA gene will provide an invaluable resource for the discovery of mutations in the gene that accounts for about 60-66% of FA patients.

Molecular characterization of Fanconi anaemia group C (FAC) gene polymorphisms.

Fanconi anaemia (FA) is a genetically heterogeneous disease with defects in at least five genes. The gene for complementation group C (FAC) has been cloned and mapped to chromosome 9q22.3 in the interval between D9S280 and D9S287. Linkage analysis is a rapid tool for the exclusion of FA families from complementation group C. The currently available markers are informative microsatellites flanking FAC and an intragenic restriction fragment length polymorphism (RFLP). In this paper, the identification of three CA polymorphic repeats localized in introns-1a, 2 and 3 and one rare variant in exon 2 are reported. The new microsatellites will enable more accurate analysis not only of FA but also in families affected by multiple self-healing squamous epitheliomata (ESS1) and nevoid basal cell carcinoma (NBCCS), since the genes of both syndromes have been mapped in the same interval as FAC.

Identification of three novel mutations in the PIG-A gene in paroxysmal nocturnal haemoglobinuria (PNH) patients.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic disorder caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins resulting from a defect in one step of GPI-anchor biosynthesis. Recent analysis has shown that mutations at the PIG-A (phosphatidylinositoglycan-class A) gene are responsible for GPI-anchor deficiency in all PNH patients. In the current study, we describe three new mutations of the PIG-A gene in Italian patients with PNH. The analysis has been performed by RNA/single-strand conformation polymorphism using genomic DNA purified from nucleated peripheral blood cells. An abnormal pattern of migration of polymerase chain reaction amplified fragments containing exons 2 and 5 was observed. Sequencing analysis led to the identification of three mutations: a transversion C-to-A creating a stop codon (Y98X), an A insertion at position 460 (460insA), and a C deletion (1114delC). All the mutations cause a premature termination of the translation of the PIG-A protein.

Characterization of the 5' region of the Fanconi anaemia group C (FACC) gene.

Fanconi anaemia (FA) is an autosomal recessive disease characterised by progressive pancytopenia, chromosome instability and an increased risk of cancer. The Fanconi Anaemia Complementation Group C (FACC) gene is mutated in patients of complementation group C. Several different forms of FACC mRNA that share the same coding region have been isolated. At least two species result from the use of alternative exons at the 5' end and three result from the use of distinct polyadenylation signals. As a first step toward the characterization of this gene we have isolated the genomic clones corresponding to the 5' region, including a putative promoter and two alternate 5' exons. These exons, named -1 and -1a, were found to be separated by a small intron, with exon -1 located 5' to exon -1a. Further, these exons are flanked by consensus sequences of donor sites at the 5' ends of introns. An acceptor splice site was not evident 5' of exon -1a, suggesting that exon -1 is not spliced onto exon -1a. The sequences upstream of exons -1 and -1a have no obvious TATA or CAAT boxes but include CG-rich sequences. Functional analysis of the sequence upstream of the putative transcription start site of both alternative exons indicates that the region upstream exon -1 is sufficient to drive the expression of the luciferase reporter gene in CaCo-2 cells and that the transcriptional regulation of this gene is complex.