Efrosini Setakis

A Genome-Wide DNA Microsatellite Association Screen to Identify Chromosomal Regions Harboring Candidate Genes in Diabetic Nephropathy

In an effort to accelerate the identification of susceptibility genes in diabetic nephropathy, the first genome-wide fluorescence-based DNA microsatellite (n=6000) association screen was performed, using pools of genomic DNA derived from Irish patients with (cases; n=200) and without (controls; n=200) type 1 diabetic nephropathy. Allele image profiles were generated for 5353 (89.2%) microsatellite markers for both case and control pools. Allele counts (estimated from allele image profiles) were compared in case versus control groups, and empirical P values were generated. Markers then were ranked on the basis of their empirical P values (lowest to highest). Repeat PCR amplification and electrophoresis of pooled samples were performed systematically on ranked markers until the 50 most associated markers with consistent results were identified. DNA samples that composed the pools then were genotyped individually for these markers. Two markers on chromosome 10, D10S558 (Pcorrected=0.005) and D10S1435 (Pcorrected=0.016), revealed statistically significant associations with diabetic nephropathy. An additional four markers (D6S281, D4S2937, D2S291, and D17S515) also are worthy of further investigation. Relevant functional candidate genes have been identified in the vicinity of these markers, demonstrating the feasibility of low-resolution genome-wide microsatellite association screening to identify possible candidate genes for diabetic nephropathy.

New candidate loci for multiple sclerosis susceptibility revealed by a whole genome association screen in a Belgian population.

We have completed a whole genome screen for association with multiple sclerosis (MS) in a Belgian population. The 6000 microsatellite markers provided through the Genetic Association of Multiple Sclerosis in EuropeanS (GAMES) collaborative were genotyped in case-control and family-based samples. The 20 most promising markers included three markers (D6S1615, D6S2444 and TNFa) from the classically established HLA class II cluster and one (D6S265) from the recently re-emphasized HLA class I cluster. In other highlighted regions, preliminary candidate genes from the immune system have been identified: e.g. the integrin ligand EDIL3, the high-mobility group box protein TOX, neutral sphingomyelinase activating factor (NSMAF) and the B-cell specific transcription factor POU2AF1.

Statistical analysis of the GAMES studies

The statistical analysis of the GAMES collaborative studies presented a number of novel challenges. Estimates of allele frequencies from pooled DNA samples are subject to additional sources of error over and above the multinomial sampling error which is assumed in classical chisquared tests for association (Barratt et al., 2002; Sham et al., 2002). Firstly, the assumption that a pool contains an equal amount of DNA from each subject will only be approximately true since quantification is subject to error. Secondly, amplification by PCR will introduce further random error. Finally, reading of concentrations of PCR products will also introduce additional errors. A fundamental difficulty is that, while reading PCR products was replicated in the design, allowing estimation of this source of error, neither pool formation nor PCR replication, was part of the standard GAMES design. It is, therefore, impossible to formally estimate the error variance attributable to each of these additional sources. For the same reason, it is not possible to estimate how the additional components of variance (and covariance) depend upon allele frequencies. As a result, formal significance tests are impossible. It will usually be argued that the purpose of DNA pooling studies is not to deliver formal tests for association, but to provide a preliminary filter to prioritize loci for later study by individual typing. For this purpose, a simple list of loci, ranked by the value of some test statistic, is all that is necessary. However, here the GAMES studies face a further difficulty as a result of the use of microsatellite markers. Such loci have a very variable number of alleles and conventional chi-squared tests for association between marker and disease state will have varying numbers of degrees of freedom. Thus, even were it not for the problems set out in the preceding paragraph, a list of markers ranked by the chi-squared for association would not be meaningful.

A genome wide scan for association with multiple sclerosis in a N. Irish case control population.

In order to screen the genome for linkage disequilibrium (LD) in multiple sclerosis (MS), we typed 2537 microsatellite markers in separately pooled DNA from 200 cases and 200 controls from N. Ireland. Twenty two markers showing significant evidence of association were identified including three from the HLA region on chromosome 6p21. Putative candidate genes mapping close to the 19 novel markers include the IL10RA and CD3E genes on 11q23 (which both lie close to the marker D11S1998). Individual typing of the marker D11S1998 confirmed its association.

Linkage disequilibrium screening for multiple sclerosis implicates JAG1 and POU2AF1 as susceptibility genes in Europeans.

By combining all the data available from the Genetic Analysis of Multiple sclerosis in EuropeanS (GAMES) project, we have been able to identify 17 microsatellite markers showing consistent evidence for apparent association. As might be expected five of these markers map within the Major Histocompatibility Complex (MHC) and are in LD with HLA-DRB1. Individual genotyping of the 12 non-MHC markers confirmed association for three of them — D11S1986, D19S552 and D20S894. Association mapping across the candidate genes implicated by these markers in 937 UK trio families revealed modestly associated haplotypes in JAG1 (p=0.019) on chromosome 20p12.2 and POU2AF1 (p=0.003) on chromosome 11q23.1.

Two genome-wide linkage disequilibrium screens in Scandinavian multiple sclerosis patients

We report the first two genome-wide screens for linkage disequilibrium between putative multiple sclerosis (MS) susceptibility genes and genetic markers performed in the genetically homogenous Scandinavian population, using 6000 microsatellite markers and DNA pools of approximately 200 MS cases and 200 controls in each screen. Usable data were achieved from the same 3331 markers in both screens. Nine markers from eight genomic regions (1p33, 3q13, 6p21, 6q14, 7p22, 9p21, 9q21 and Xq22) were identified as potentially associated with MS in both screens.

A whole genome screen for linkage disequilibrium in multiple sclerosis performed in a continental Italian population

We have systematically screened the genome for evidence of linkage disequilibrium (LD) with multiple sclerosis (MS) by typing 6000 microsatellite markers in case-control and family based (AFBAC) cohorts from the Italian population. DNA pooling was used to reduce the genotyping effort involved. Four DNA pools were considered: cases (224 Italian MS patients), controls (231 healthy Italians), index (185 index cases from trio families) and parents (the 370 parents of the patient included in the Index pool), respectively. After refining analysis of the most promising 14 markers to emerge from this screening process, only marker D2S367 retained evidence for association.

A genome-wide screen for linkage disequilibrium in Sardinian multiple sclerosis

Using indirect whole genome association screening, we have searched for multiple sclerosis susceptibility genes in the genetically isolated high risk Sardinian population. Two screens were performed; the first was based on 229 cases and 264 unrelated controls, and the second on 235 trio families. Each screen employed a dense set of microsatellite markers and DNA pooling. Data from both screens were available from 2764 markers. Nine markers showed nominally significant results in both screens independently. Five of these markers-D2S408 (2q36), D6S271 (6p21), D6S344 (6p25), D7S1818 (7p12) and D16S420 (16p12)-remained nominally significant in both studies after conservative refining analysis.

A GENOME-WIDE GERMAN SCREEN FOR LINKAGE DISEQUILIBRIUM IN MULTIPLE SCLEROSIS

We report on a genome-wide screen for association with multiple sclerosis (MS) in the German population performed using 6000 microsatellite markers. These markers were typed in four DNA pools consisting of 234 MS patients (cases), 209 unrelated controls, 68 index patients from trio families and their 136 parents (related controls). Stringent analysis identified 11 markers showing apparent evidence for association. Five from regions previously identified in linkage studies and two from the MHC region on chromosome 6p21. These MHC markers are known to be in linkage disequilibrium with HLA class II alleles influencing susceptibility to MS. The identification of these markers serves as an important positive control.

A genome-wide screen for association in Hungarian multiple sclerosis

Although the pathogenesis of multiple sclerosis (MS) is not fully understood, substantial evidence points to the involvement of genetic factors. We report on a genome-wide screen for disease association in the Hungarian population using 5532 microsatellite markers. These markers were typed in DNA pools that consisted of 88 MS patients (cases), and 128 unrelated controls. Based on a stringent selection criterion, we obtained 33 markers suggesting association with the disease.