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Francisco
E Baralle, ICGEB Trieste Italy
Franco Pagani, ICGEB Trieste Italy
Summary:
When sequence variants are identified in genomic DNA, especially for routinely sequenced disease-associated genes, the correct interpretation of the molecular nature of the substitution may not be immediately evident. The effect of the mutations on gene expression is generally assumed to depend on its location. Because exonic mutations are widely believed to cause disease by modifying the coding composition of the sequence, silent mutations have been ignored as potential cause of disease. Missense mutations have been assumed to produce significant alterations only in protein function and intronic variants, if not at the splice sites, are largely ignored. Recent evidences from many laboratories have now identified that the primary mechanism of disease in a significant fraction of disease-causing mutations is a catastrophic splicing abnormalities that disrupt unrecognised splicing regulatory elements. SNPs (Single Nucleotide Polymorphisms) in each of these novel exonic or intronic sequences can cause pathological splicing events. Thus, aberrant splicing patterns may result from disruption of complex splicing regulatory mechanisms for which the basic molecular events are still greatly unknown and their effect on splicing is not obvious.
This workshop will
focus on an integrated view for a comprehensive understanding and management
of pre-mRNA splicing defects in human pathology. The workshop will connect basic
research in different pre-mRNA processing steps with human genetics where there
are frequent observations of "orphan" DNA mutation/variant without
a clear functional role in the processing of mRNA. Frequently these mutations
are found both in patients with the disease or in non-affected individuals,
or are found in patients with variable disease severity. The workshop would
promote collaboration among the expertise and scientific competence of research
laboratories working in different steps of the pre mRNA processing factory with
diagnostic and human genetic screenings laboratories.
Topics will include basic mechanism of pre-mRNA processing, as for example the
role of splicing factors in regulating the alternative splicing and the definition
of new splicing regulatory elements, new methods for the screening and functional
identification of pre-mRNA processing defects in human pathology and new strategies
for the correction of the processing defects. The integration of basic research
and human genetic laboratories will have an impact in the understanding of the
phenotypic variability due to pre-mRNA processing defects which potentially
covers several human diseases. This workshop would be unique to connect basic
research in the functional genomic area of pre-mRNA processing, which is a fast
developing research topic, with human molecular diagnostic and screening laboratories.
Background:
Splicing defects are quite common in human disease. A survey performed more then a decade ago found that about 15% of point mutations disrupted splicing 1. However this is an underestimation because the analysis was limited to mutations in the classical splice site sequences. Extensive characterization of mutations in the neurofibromatosis (NF1) gene and ATM gene demonstrated recently that 43-48% of these mutations cause splicing alterations 2,3. The simple presence of basic splicing elements is not sufficient to explain the complexity of the exon splicing selection. In fact the process of exon recognition during pre mRNA processing occurs through a complex network of multiple interactions. A number of cis-acting elements variably distributed in pre mRNA (splicing enhancers and silencers) and trans-acting factors (SR proteins, hnRNPs and snRNAs), operates in a tissue specific manner to regulate the constitutive and alternative splicing of different exons 4. Regulatory splicing factors are considered to interact with component of the basic splicing machinery and recognize splicing elements (enhancer and silencer) within the exon. In addition, recent evidences indicate that several multi-component cellular machines that carry out separate steps in the gene expression pathway, which include transcription, several pre-mRNA processing steps and the export of mature mRNA to the cytoplasm are interfaced both physically and functionally 5. During transcription the nascent pre-mRNA is capped, introns are removed by splicing and the 3' end is polyadenylated. The mature mRNA is then released from the site and exported to the cytoplasm for translation and the RNA surveillance system eliminate aberrant mRNAs. The comprehension of the coupling among these different gene expression machines is important in order to understand the genome diversity and the effect of gene mutations/variations in human disease.
Given the complexity of the splicing machinery, when sequence variants are identified in genomic DNA, especially for routinely sequenced disease-associated genes, the correct interpretation of the molecular nature of the substitution may not be immediately evident. The identification of disease causing mutations is based primarily on linkage of the mutation with the disease phenotype. The effect of the mutations on gene expression is generally assumed based on its location. Because exonic mutations are assumed to cause disease by affecting only the coding potential, silent mutations have been ignored as potential cause of disease, missense mutations have been assumed to create a significant alterations in protein function, and nonsense mutations have been assumed to lead to expression of non functional or deleterious truncated proteins or loss of function from nonsense mediated decay. Recent evidences from many laboratories have now identified that the primary mechanism of disease in a significant fraction of disease-causing exonic mutations is a catastrophic splicing abnormalities that disrupt unrecognised splicing regulatory elements 6. The occurrence of single base changes in each of these novel exonic or intronic sequences can cause pathological splicing events by inducing exon skipping or aberrant exon inclusion 7-12 The same situation may also apply for common genomic variants reported in human association studies. In this case, the assumption is that some of these genetic polymorphic variants might contribute significantly to genetic risk for common diseases, as they can be the etiologic cause of disease-susceptibility differences. The identification of these new disease causing mechanism that affect splicing has led recently to the development of new therapeutic strategies to correct the splicing defects 13-15
Topics:
The workshop will be held at the International Centre for Genetic Engineering and Biotechnology,
Trieste, Italy.Eligibility for participantion:
Participants must have a degree in medicine or biology, and working in medical genetics or functional genomics. Priority will be given to participants coming from countries that financially support the European Science Foundation (ESF) and from ICGEB member states. Preference will be given to young scientists and the final numbers for the meeting are limited to 60 maximum.
Dates: April 7-9, 2005
Programme:
(provisional)
Day 1
Introduction to basic mechanism of pre-mRNA processing - Benoit Chabot
Control of alternative splicing - Adrian Krainer
Cis and trans acting factors involved in pre mRNA splicing - Livio Pellizzoni
Pre mRNA structure and exon definition - Juan Valcarcel
Coupling of transcription and RNA processing - Nick Proudfoot
Day
2
5' and 3' splice site selection and splicing defects - Thomas Cooper
Splicing defects at exonic cis-acting elements - Javier Caceres
Splicing defects at intronic cis-acting elements - Benoit Chabot, Juan Valcarcel
Promoter architecture and splicing variations - Franco Pagani
Pre mRNA splicing defects, selected examples 1 - Livio Pellizzoni (spinal muscular
atrophy, Thomas Cooper (Neurological Diseases),
Day
3
Pre mRNA splicing defects, selected examples 2 - Francisco Baralle (Cystic Fibrosis,
Neurofibromatosis), Thilo Doerk (ATM)
Methods for the screening and functional identification of pre-mRNA processing
defects - Chris Smith
Strategies for the correction of the processing defects, ESSENCE compounds,
modified snRNAs - Adrian Krainer
Interactive sections will be organised during the meeting.
Pre mRNA processing: distinguishing between benign and disease-causing sequence
SNPs.
Methods to identify pre mRNA processing defects, in vitro splicing, the hybrid
minigenes and computer assisted methods
Cases presented by the participants
Registration is now closed.