Most complex diseases are the result of interactions
between genetic susceptibility and exposure to a vast array of environmental,
dietary and endogenous factors. Classical methods that distinguish the contributions
of nature and nurture-concordance rates in twins, adoption studies and pedigree
analysis have shown that many complex diseases are strongly influenced by genes.
The genetic component is most likely heterogeneous, with multiple genes having
modest independent and interactive influences on the overall disease phenotype.
In fact, for genetically complex diseases, some authors have outlined the "common
disease-common variant" hypothesis, which states that common disease susceptibility
or resistance variants are expected to be few at each locus, relatively common
in the human population and enriched in the coding and regulatory sequence of
genes (Chakravarti et al., 1999). An important new tool that is expected to
contribute to the deciphering of common complex disorders is the use of single
nucleotide polymorphisms (SNPs). SNPs are common inherited sequence polymorphisms
found frequently (at intervals of roughly every 300-500 bp) throughout the human
genome and could contribute significantly to the genetic risk of common diseases.
Several studies have investigated the association between single or few SNPs
and some complex diseases, but is now emerging a new genetic approach using
gene-based haplotype which is predicted to have more power than individual SNP
to track the disease causative site (Judson et al., 2000). Functional studies,
performed by Workpackage 3, on putative causative SNP will then verify if it
has small quantitative effect on disease susceptibility.
In order to refine and focus our programme goals, we decided to focus our attention
on only four complex diseases: inflammatory bowel disease, multiple sclerosis,
chronic obstructive pulmonary disease and Type 2 Diabetes.
Brief genetic background of these diseases is here reported:
Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation. IBD is subdivided in Crohns disease and ulcerative colitis phenotypes. Both diseses have a combined prevalence of 100 to 200 per 100,000 in the Western World. The etiology of IBD is unknown. Association studies have been focused on genes involved in the regulation of the immune response (HLA-, TNF-, IL-1RA, ICAM-1 genes. Genome scanning have been employed to map some susceptibility locus on chromosomes 16 (IBD1), 12 (IBD2), and 6 (Yang & Rotter,2000). Recent studies have demonstrated that IBD is characterized by an imbalance of Th1/Th2-cells and interleukin-12 (IL-12), composed of a p40 and p35 subunits, plays a pivotal role in Th1 mediated immune response and tissue injury (Pallone et al., 1998). It has been observed also increased levels of IL-12 transcripts for both IL-12-subunits (p35 and p40). However, the molecular basis of genetic regulation of IL-12 responsiveness in IBD is not completely understood. Moreover, Pravica et al.,(2000) recently describes rare polymorphisms in the promoter regions of the IL-12 p35 and p40 subunits.
Type 2 diabetes mellitus is the most common form of diabetes worldwide,affecting approximately 4% of the world's adult population. It is multifactorial in origin with both genetic and environmental factors contributing to its development. Genome-wide screens have lead to the localization of susceptibility genes for type 2 diabetes to different regions of the genome, suggesting that different combinations of susceptibility genes contribute to the development of type 2 diabetes (Taylor et al., 1999). Recently, has been cloned, by positional cloning, a gene in the NIDDM1 region on chromosome 2, that shows association with type 2 diabetes. This gene encodes the cysteine protease calpain-10 (Horikawa et al., 2000).
Multiple sclerosis is an inflammatory demyelinating disease that is thought to share a dependence upon an autoimmune response to components of the myelin sheath. The prevalence varies considerably around the world, ranging from 5 in 100,000 to more than 30 in 100,000 persons. Evidence that genetic factors have a substantial effect on susceptibility to multiple sclerosis is unequivocal. In 1973 it has been recognized that the presence of the HLA-DR2 allele substantially increases the risk of multiple sclerosis. Wide genome scans have been performed and regions of interest have been identified, although none have been linked to the disease with certainty. Recently, variants of the interleukin-1(beta)-receptor and interleukin-1-receptor antagonist genes, immunoglobulin Fc receptor genes and apolipoprotein E gene have been associated with the course of the disease, but these findings await confirmation. Overall, it seems likely that MS is marked by considerable genetic heterogeneity and that no strongly predisposing alleles are widely prevalent (Noseworthy et al., 2000). As reviewed by Borsellino et al. (2000), autoimmune processes in chronic MS tissues are mediated by gd T cells, and these have been shown to respond to non-peptide antigens in an MHC independent manner. Several studies suggested that glycolipids presented through members of the CD1 family of MHC class I related molecules could be involved in this process. Since lipids are a major component of the myelin sheath it is reasonable to consider the possibility that nervous system lipids might be involved in the immunopathogenesis of demyelinating disorders. Based on this data, CD1b gene (Battistini et al., 1996) could be considered a candidate gene, not yet genetically investigated, for MS.
Chronic obstructive pulmonary disease (COPD) is characterised by chronic airflow limitation which progresses slowly over a period of years and is largely irreversible; its primary pathology is pulmonary emphysema. A number of studies indicate that genetic factors contribute to the risk of COPD (Koyama & Geddes, 1998). It has been hypothesised that oxidant-antioxidant imbalance and protease-antiprotease imbalance may be important in its pathogenesis. Variants of the GST M1, GST P1, heme oxygenase-1 gene promoter polymorphisms, mEPHX and a1-antitrypsin gene polymorphisms have been associated with an augmented risk to develop COPD (Yamada et al., 2000; Koyama & Geddes, 1998). Increased CFTR gene mutations have been found in patients suffering from chronic bronchitis (Kostuch et al., 2000).
· Chakravarti A. Population genetics-making
sense out of sequence. Nat Genet 1999, 21(1Suppl):56-60.
· Judson et al., The predictive power of haplotypes in clinical response.
Ashley Publications 2000, 1:5-16.
· Yang &Rotter. The genetic background of inflammatory bowel disease.
Hepatogastroenterology 2000, 47(31):5-14.
· Pallone et al., Interleukin-12 and Th1 response in inflammatory bowel
diseases. Gut 1998, 43:735-736.
· Pravica et al. Rare polymorphisms in the promotor regions of the human
interleukin-12 p35 and interleukin-12 p40 genes. Eur J Immunogenet 2000, 27:
35-6.
· Noseworthy et al., Medical progress: Multiple Sclerosis. N Engl J Med
2000, 343(13).938-952.
· Borsellino et al., J Neuroimmunol 2000, 107:124-129.
· Koyama &Geddes. Genes, oxidative stress, and the risk of chronic
obstructive pulmonary disease. Thorax 1998, 53 (Suppl 2):S10-S14.
· Yamada et al., Microsatellite polymorphism in the heme oxigenase-1
gene promoter is associated with susceptibility to emphysema. Am J Hum Genet
2000, 66:187-195.
· Kostuch et al., Detection of CFTR gene mutations in patient suffering
from chronic bronchitis. Arch Med Res 2000, 31(1):97-100.
· Taylor et al., Deconstructing Type2 diabetes. Cell 1999, 97: 9-12.
· Horikawa et al., Genetic variation in the gene encoding calpain-10
is associated with type 2 diabetes mellitus. Nat Genetics 2000, 26(2): 163-175.
The workpackage's objective is to set up a reliable and reproducible test for
variant identification and screening in complex disease candidate genes. The
achievement of this purpose is important to constitute a subset of putative
causative DNA variants to be studied functionally by workpackage 3.
In this main objective some intermediate steps can be identified, such as:
- Identification of functional genomic variants (SNPs);
- set up of experimental condition of PCR and DHPLC analysis;
- generation and isolation of high quality, full-length coding sequences from
information contained in Type 2 diabetes-specific expressed sequence tags (ESTs)
(specific objective of research line 2)
- creation of a DNA/RNA bank from patients affected by inflammatory bowel disease,
multiple sclerosis, chronic obstructive pulmonary disease and Type I diabetes
The workpackage is organised in two research line which are interactive and
are described in the appropriate section.