Gene Expression Profiling As a Tool for Positional Cloning of Genes- Shortcut Or the Longest Way Round
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494 Current Genomics, 2008, 9, 494-499 Gene Expression Profiling as a Tool for Positional Cloning of Genes- Shortcut or the Longest Way Round Lena W. Rosenlöf * Section for Immunogenetics, University of Rostock, Germany Abstract: The identification of quantitative trait loci, QTL, in arthritis animal models is a straight forward process. How- ever, to identify the underlying genes is a great challenge. One strategy frequently used, is to combine QTL analysis with genomic/proteomic screens. This has resulted in a number of publications where carefully performed genomic analyses present likely candidate genes for their respective QTL´s. However, seldom the findings are reconnected to the QTL con- trolled phenotypes. In this review, we use our own data as an illustrative example that “very likely candidate genes” iden- tified by genomic/proteomics is not necessarily the same as true QTL underlying genes. Received on: June 18, 2008 - Revised on: June 23, 2008 - Accepted on: June 28, 2008 1. INTRODUCTION healing arthritis, but some models e.g. autologous collagen II induced arthritis, aCIA, and pristane-induced arthritis, PIA, Despite last decade’s therapeutic progression, Rheuma- in rats [5] are also suitable for studies on chronic relapsing toid arthritis (RA) still lacks cure and satisfactory treatment arthritis. In resemblance to RA these models are highly de- in many cases. To make a change, it is urgent to elucidate the pendent on the genetic background, with MHC as the most underlying disease mechanisms. As RA has a genetic com- important individual region, but with obvious contribution ponent of ~60% [1], one way to go is to identify predispos- also of other genes [5, 6]. The assumption is that these other ing genes. The strongest genetic contribution to RA comes genes also need to be identified in order to understand RA. from the HLA region, which is expected to count for about To do so, a first step is to identify chromosomal loci control- 35% of the genetic predisposition [2]. However, to learn ling quantitative arthritic traits (quantitative trait loci, QTL). more about underlying mechanisms also additional contrib- This is done with genome-wide linkage analyses in crosses uting genes need to be identified. Unfortunately, this has between susceptible and resistant strains. Nowadays, QTL turned out to be far from easy as the genetics behind RA is identification is pretty strait forward and ~70 joint/bone in- complex, i.e. heterogeneous, polygenetic with low pene- flammation QTL´s have been identified for the rat [7] and trance of each contributing gene, epistatic and with interac- almost 400 arthritis QTL´s for the mouse [8]. However, the tion between genes and environment. As the human popula- subsequent step, to identify the underlying gene(s), has tion is outbred and exposed to highly variable environment, turned out to be challenging. So far, only a small number of it is really difficult to reproducibly, identify predisposing genes have been identified from a QTL and then it has been genes in human material. Also, phenotypes necessary for the genes from QTLs that have extraordinary large effect sizes understanding of disease mechanisms (e.g. disease on-set [9]. and progression from acute to chronic inflammation) cannot be studied in humans as most patients once they get their 3. STRATEGIES FOR GENE CLONING FROM A QTL diagnosis already are in a chronic state of disease. In an at- tempt to overcome these problems, we and others use animal Before consider strategies for cloning a gene from a models. Animals can be genetically and environmentally QTL, one has to be aware of what one is looking for. As has controlled, genetically manipulated and the disease can be been nicely pointed out by Flint et al. [9] most single nucleo- followed in detail before on-set until the progression into tide polymorphisms, SNPs, underlying QTL´s for complex chronic disease. diseases are not likely to be found within coding-sequences. In the worse case the position of the quantitative trait nucleo- 2. FIRST STEP OF GENE IDENTIFICATION –QTL tide, QTN does not coincide with the position of the gene it IDENTIFICATION is affecting (trans-regulated genes). However, let us assume that the causative variant of a gene indeed is encoded from Firstly, to identify RA relevant genes in rodents, a dis- the QTL. Then, to work out strategies for its identification it ease model with high similarity to the human disease has to is important to have in mind which criteria such a gene be chosen. Several such models have been developed; e.g. should fulfill. Suggested criteria are; the gene should be po- induction by injection of cartilage proteins in adjuvant oils lymorphic in a way that leads to altered protein product or [3] or by injection of adjuvant oils only (in rats) [4]. A draw- differential expression, its expression should be detected in back of some models is that they only result in acute self- organs or cell types of relevance and the gene shall have a function either related to the trait directly or in a biologic *Adress correspondence to this author at the Section for Immunigenetics, pathway of relevance for the trait [10, 11]. However, a typi- University of Rostock, Schillingallée 70, 180 55 Rostock, Germany; E-mail: [email protected] cal QTL identified in a F2-cross encodes too many genes to 1389-2029/08 $55.00+.00 ©2008 Bentham Science Publishers Ltd. Gene Expression Profiling as a Tool for Positional Cloning Current Genomics, 2008, Vol. 9, No. 7 495 allow criteria fulfillment analysis of all the genes. Therefore, with bioinformatics tools have been suggested in [20]. This the QTL need to be narrowed to reduce the possible candi- includes comparative genomics, combined cross analysis, dates to a number that can be handled for investigation. To interval specific haplotype analysis, sequence comparison narrow a QTL, one can choose between safe, “hard-core and expression data base analysis. evidence” strategies and “likelihood” based strategies. The An intermediate strategy effort and economically wise, is former are often expensive, laborious and time-consuming to make congenes and use genomic and proteomic screens to sometimes even beyond the border of be performable at all. narrow the QTL. This strategy is aimed to, in a nearly unbi- The second strategy is not so safe but can more easily be ased way, find genes within the QTL that fulfills the differ- afforded, economically as well as time- and effort-wise. ential qualitative protein product and differential expression 3.1. Safe, “Hard-Core Evidence” Strategies criteria for QTL underlying genes. This strategy have gained popularity after some nice publications showing that the The classical way of gene identification is to construct a strategy indeed works and gives candidate genes that can be congenic strain where the QTL region of the F2-cross paren- referred back to the QTL phenotype [21-24]. In the light of tal strains “A” has been substituted with the corresponding this we also tried this strategy with our QTL Pia6 that spe- region from parental strain “B”. This is done by systematic cifically control the chronic phase of PIA. breeding for 6-10 generations depending on method [12], to receive a” B”- derived QTL genomic fragment on a pure “A” 4. BACKGROUND OF THE DA.PIA6 CONGENE background genome. If the phenotype can be reproduced in the congenic animal, one can conclude that the affecting The PIA6 QTL was identified in an F2-cross between the polymorphism indeed is encoded from this region. To nar- PIA susceptible DA and PIA resistant E3 rat [6]. The region row the causative fragment using the congenic animals, the was specifically associated with arthritis chronicity increas- animals are further backcrossed and genotyped. Off-springs ing in importance over time (no of affected paws day83, where a genomic recombination has occurred, resulting in a LOD score 4.2 and day 120, LOD score 4.9). The inheritance smaller “B” –derived congenic fragment, are tested for the was DA recessive with E3 as the protective parental. The disease phenotype. Congenics positive for the phenotype are variance explained by the locus was 16-19% and the peak selected for further backcross. This method is safe when sys- marker was D14Csna (21.9 MBp) and flanking markers were tematically performed and has successfully been used in approximately at 15 MBp and 45 MBp (positions according cloning of the arthritis susceptibility gene Ncf1 [13]. How- to reference sequence, NCBI). We produced a congenic rat, ever, to be feasible, as discussed below, it assumes that the DA.Pia6 with speed congenic technique. A ~50Mp fragment QTL underlying gene has a strong, dominant, sex-indepen- between D14Wox8 (19.1 MBp) and D14rat64 (68.6 MBp) dent effect on the phenotype. In addition, this strategy is time from the E3 rat was introgressed on a DA background. This consuming as it is dependent of recombinant events to occur, congenic rat was shown to specifically protect from chronic which cannot be forced. The only possibility to speed up the arthritis [25]. Using this rat, the underlying gene should be procedure is to provide resources for a large breeding and possible to clone and generate essential knowledge of simultaneous phenotype-testing of many subcongenics. An- chronic inflammation specific processes. other strategy to collect recombination is to produce ad- Previously, in our lab, it was shown that a gene, Ncf1, vanced intercross lines (AIL) [14]. In these lines recombina- underlying a QTL Pia4 indeed could be positionally cloned tions are collected by continue the intercross several genera- using a strategy of recombinant selection where the disease tions beyond F2. AIL´s has been successfully used to narrow phenotype was used as selection criteria [13].