|Home | About | Journals | Submit | Contact Us | Français|
Previously, an analysis of 14 extended, high-risk Utah pedigrees localized the chromosome 22q linkage region to 3.2 Mb at 22q12.3-13.1 (flanked on each side by three recombinants), which contained 31 annotated genes. In this large, multi-centered, collaborative study, we performed statistical recombinant mapping in fifty-four pedigrees selected to be informative for recombinant mapping from nine member groups of the International Consortium for Prostate Cancer Genetics (ICPCG). These 54 pedigrees included the 14 extended pedigrees from Utah and 40 pedigrees from eight other ICPCG member groups. The additional 40 pedigrees were selected from a total pool of 1,213 such that each pedigree was required to both contain at least four prostate cancer (PRCA) cases and exhibit evidence for linkage to the chromosome 22q region. The recombinant events in these 40 independent pedigrees confirmed the previously proposed region. Further, when all 54 pedigrees were considered, the three-recombinant consensus region was narrowed by more than a megabase to 2.2 Mb at chromosome 22q12.3 flanked by D22S281 and D22S683. This narrower region eliminated 20 annotated genes from that previously proposed, leaving only eleven genes. This region at 22q12.3 is the most consistently identified and smallest linkage region for PRCA. This collaborative study by the ICPCG illustrates the value of consortium efforts and the continued utility of linkage analysis using informative pedigrees to localize genes for complex diseases.
The International Consortium for Prostate Cancer Genetics (ICPCG) was created to foster collaborative efforts towards gene identification through powerful, combined analyses using linkage data in pedigrees. Currently, the ICPCG brings together genome-wide linkage data from eleven international prostate cancer (PRCA [MIM 176807]) research groups comprising 1,310 pedigrees. The initial ICPCG genome-wide linkage scan for PRCA identified chromosome 22q with the strongest linkage evidence (LOD score of 3.57) with a 1-LOD support interval from 35 to 47 cM (1). This combined linkage analysis used a strict definition for PRCA and particular linkage techniques which, in some cases, imposed restrictions on pedigree structure. Several of the individual groups’ linkage studies, which often differed in disease definition and linkage method, also reported linkage evidence to the 22q region with varying degrees of significance (2–9). This makes chromosome 22q the most consistently identified linkage region for PRCA genes.
Localization efforts are challenging, in particular due to genetic heterogeneity; statistical noise from unlinked pedigrees and phenocopy PRCA cases can misleadingly shift linkage evidence. One approach for localization is therefore to focus on only those pedigrees that are unlikely to represent mere chance clustering and that contribute most to the overall linkage signal. That is, multi-case pedigrees that exhibit at least nominally significant linkage evidence to a region of interest. The disadvantage of this approach is that only a few such pedigrees may exist in a single collection. However, such an approach may be possible in an international collaborative setting, such as the ICPCG.
Camp et al (10) performed recombinant mapping in 14 high-risk, extended Utah pedigrees selected from their previous genome-wide linkage study (7) to localize the 22q region to 3.2 Mb at 22q12.3-13.1 containing 31 known genes. In the present collaborative study, we describe further localization of the region using data from an additional 40 pedigrees each with multiple PRCA cases and at least nominally significant evidence for linkage to the chromosome 22q region.
From the total of 1,213 ICPCG pedigrees, forty were identified with at least four confirmed PRCA cases and a pedigree-specific LOD score indicating at least nominal evidence for linkage to the chromosome 22q region. Recombinant mapping was performed for each of these forty pedigrees, which entails using the LOD graphs to identify the recombinant positions and identify the shared, linked, segregating haplotype. Figure 1 shows an example of a LOD graph for a single pedigree (Michigan pedigree 6196). A single recombinant has occurred between D22S315 at 22.88 cM (where the LOD score is >1.0) and D22S539 at 14.68 cM (where the LOD score is <−0.5). The true position of the recombinant could lie anywhere between these two markers. We use the outermost marker to define the recombinant position. This is the conservative approach because it can only over-estimate the shared chromosomal segment and hence the resulting consensus region. As shown in Figure 1, the most conservative estimation for the position of the recombination in Michigan-6196 is at D22S539. The linked haplotype is therefore recorded from D22S539 to the q-terminus. We performed this for each pedigree and overlaid the haplotype segments to identify the consensus region across all pedigrees. In particular, the consensus region flanked by three recombinants on each side is akin to a 99% credible interval (see Methods section).
Figure 2 shows the results from the recombinant mapping. The non-recombinant chromosomal segments, both for the additional 40 ICPCG pedigrees and for the 14 Utah pedigrees which initially localized the region are shown (10). The previously described three-recombinant region was at chromosome 22q12.3-13.1 and stretched from D22S281 at 38.55 cM (32,666,708 bp) to D22S1045 at 44.75 cM (35,866,231 bp). In the 40 pedigrees in the current study, the three-recombinant region is flanked by D22S280 at 37.03 cM (31,295,286 bp) and D22S283 at 41.63 cM (35,080,651 bp). Considering all 54 pedigrees together, the consensus one-recombinant region remains between D22S1265 at 39.06 cM (33,719,908 bp) and D22S277 at 40.82 cM (34,601,466 bp). The consensus three-recombinant region is narrowed to 2.2 Mb at 22q12.3 between D22S281 at 38.55 cM (32,666,708 bp) and D22S683 at 41.23 cM (34,843,637 bp). This represents a reduction of 30%, or 1,022,594 bp, from that reported previously, and eliminates 20 genes.
Analysis of these 40 ICPCG pedigrees not only corroborates the region previously described, but provides a more precise definition of the 22q region containing the hypothesized susceptibility variant(s). Figure 3 shows a schematic indicating the location of the 11 known genes within the 2.2 Mb consensus three-recombinant region at chromosome 22q12.3 (ISX, HMG2L1, TOM1, HMOX1, MCM5, RASD2, MB, LOC284912, APOL6, APOL5 and RBM9).
We have analyzed 40 ICPCG pedigrees, each with at least four PRCA cases confirmed by medical record or death certificate and with pedigree-specific linkage evidence to the region to further localize the PRCA linkage evidence on 22q. Together with the 14 high-risk Utah pedigrees that were used to perform the initial localization, the total collection of 54 pedigrees contributes 55 defining recombinant events that taken together provide identification of a consensus region with no conflicts. The one-recombinant consensus region is an 881,538 bp interval containing eleven genes. More conservatively, if we consider the putative 22q12.3 PRCA locus as the larger three-recombinant region, the physical size is 2.2 Mb, but contains no additional known genes. In particular, the two additional telomeric recombinants provided by ICPCG pedigrees analyzed here narrowed the previously proposed (10) three-recombinant region by over 1 Mb, removing 20 genes from the region. No further narrowing was achieved on the centromeric end of the region; however, there is a gene desert in this direction and no genes lie centromeric of D22S1265.
This study illustrates the utility of statistical recombinant mapping to localize linkage regions. In this particular study, the 1-LOD support interval from the collaborative genome-wide scan was a 12 cM (approximately 8 Mb) region (1). However, the three-recombinant consensus region using the same data (without Utah) in a pedigree-specific manner is a 4.6 cM (3.8 Mb) region. Note that this region size is smaller than the genomic search resolution of any individual group’s map. This “fine-mapping” is achieved simply by virtue of the use of different marker maps by the different groups. Incorporating the Utah pedigrees with their fine-mapping markers, the region becomes 2.7 cM (2.2 Mb). If fine-mapping markers were to be added to select ICPCG pedigrees, there would be potential to narrow the region even more.
The individual pedigree-based strategy for localization seems to have been largely overlooked for mapping complex disease genes but, through consortia efforts, it may prove beneficial in localizing linkage regions and moving towards successful gene identification. The key undoubtedly lies in the ability to identify multiple, informative localization pedigrees to provide the statistical recombinant events. The requisite properties of such pedigrees include a significant excess of disease to avoid studying pedigrees which are merely chance clustering of disease and good evidence for segregation of the region of interest with the disease. Here we focused on only those pedigrees that contained at least four PRCA cases and that had a nominally significant pedigree-specific LOD score (LOD≥0.588; p≤0.05). We considered these were reasonably strict criteria, which were satisfied by 40 pedigrees from the total resource of 1,213 (3%). In addition to disease clustering and linkage evidence, another valuable characteristic for a localization pedigree is having a large number of meioses (gained via large numbers of generations and/or large sibships). It is clear that the more meioses contained in a pedigree, the more opportunities to observe recombinant events, although the disadvantage with a large numbers of generations is the increased likelihood of intra-familial genetic heterogeneity. In fact, the proportionately larger number of meioses in the high-risk Utah pedigrees is likely the reason that a single research group was able to perform the initial localization effectively (10). On average 1.5 times more recombinant events were observed across chromosome 22 in the Utah pedigrees than in the pedigrees from other groups.
The fundamental advantage of the ICPCG is that through collaborative efforts, the consortium creates a large pool of pedigrees from which homogeneous subsets can be selected for specific purposes, such as in the current study or in the ICPCG aggressive disease analysis (11). But more than this, it also brings together a diversity of ethnicities and origins, ranging from Northern European pedigrees from Finland to African American pedigrees in the AAHPC. Inspecting results across this spectrum may be informative. In the current study, Table 2 indicates the proportion of powerful pedigrees (with ≥ 4 PRCA) that were found to be linked (LOD ≥ 0.588) to the chromosome 22q12 region. The proportion of informative linked pedigrees calculated across all groups was 0.07 (40/538), but proportions ranged from 0.00 (0/37) in German pedigrees collected at University of Ulm to 0.16 (7/45) in the University of Michigan resource. However, even these two extremes are not statistically different than the global estimate across all pedigrees (p>0.05). Thus, while the underlying gene at chromosome 22q12 may only account for less than 10% of pedigrees, it does seem reasonably consistent across populations.
Multiple studies have suggested that a PRCA susceptibility gene resides in the chromosome 22q12 region (1–9). Through analysis of 54 selected pedigrees we have used recombinant mapping to narrow the consensus three-recombinant region to 2.2 Mb at 22q12.3 between D22S281 and D22S683. There remains only a small probability (p<0.007) that the putative disease gene lies outside this region. In addition, we have established that the 881,538 bp interval, between D22S1265 and D22S277 remains the consensus one-recombinant region, which is the most likely to contain the 22q PRCA predisposition gene. This collaborative study from the ICPCG illustrates the strength of the consortium and indicates the continued utility of linkage analysis in informative pedigrees to localize genes for complex diseases.
The ICPCG comprises 1,310 pedigrees with genomic search data: 1,233 pedigrees from ten groups (referred to as ACTANE, BC/CA/HI, Johns Hopkins University, Mayo Clinic, Fred Hutchinson Cancer Research Center (FHCRC), University of Michigan, University of Tampere, Finland, University of Ulm, Germany, University of Umeå, Sweden, and University of Utah) that are described in detail elsewhere (12) and 77 pedigrees from the African American Hereditary Prostate Cancer (AAHPC) group (9). For the chromosome 22 region, the initial localization efforts were carried out as an independent study of high-risk Utah pedigrees without limitations on pedigree structure (10). Removing the Utah component from the ICPCG, the remaining consortium total is 1,213 pedigrees with genomic search linkage data. Here we concentrate on the additional localization information found using these 1,213 pedigrees. All linkage results presented for the pedigrees are based on a strict PRCA definition (confirmed by medical record or death certificate) and parametric multipoint LOD scores calculated using the Genehunter-Plus software (13), and utilizing the ‘Smith’ model (14) which was used in the original ICPCG genome-wide linkage scan that indicated the linkage to the region on 22q (1). This model assumes a sex-limited, dominant gene with a disease allele frequency of 0.003 and a 15% phenocopy rate. Unaffected men under 75 and women are of unknown phenotype. In unaffected men over 75, a penetrance for carriers of 63% was assumed, while the risk for non-carriers was 16%. Linkage evidence was calculated at a 1 cM resolution across the chromosome for each pedigree. The genetic markers available for each group and the consensus map used are shown in Table 1. The research protocols and informed consent procedures were approved by each group’s institutional review board.
Table 2 shows pertinent characteristics of the pedigrees from the ten ICPCG groups. Localization pedigrees were chosen to be those with at least four confirmed PRCA cases and with LOD ≥ 0.588 (p ≤ 0.05) within the 1-LOD support interval (35 – 47 cM) previously described by the ICPCG genome-wide linkage scan (1). These represent the criteria used in the initial localization of Camp et al (10). A total of forty pedigrees satisfied these criteria, comprising five pedigrees from the AAHPC group, one pedigree from the ACTANE group, one pedigree from the BC/CA/HI group, fourteen pedigrees from Johns Hopkins, five from Mayo Clinic, six from the FHCRC, seven from the University of Michigan and one from the University of Umeå in Sweden.
For each pedigree with a LOD ≥0.588, the multipoint LOD graph was used to estimate the position of recombinant events. A sharp decrease in LOD (>0.5 LOD units) was used to indicate loss in sharing (recombination). To remain conservative, we report the outermost possible position of each recombinant. For each pedigree, the non-recombinant chromosomal segments are overlaid to create the consensus region. The minimal one-recombinant consensus region is the region defined by one recombinant at each side. Similarly, two- and three-recombinant consensus regions can be defined. In particular, we focus on the three-recombinant consensus region because, even if we consider a phenocopy PRCA rate as high as 15%, the probability that the gene lies outside of the region is very low (probability = 0.007). That is, the putative mutation will lie outside of the region only if all three boundary recombination events were observed, by chance, in phenocopy PRCA cases, on either or both sides of the region, which is simply 2×(0.15)3 − (0.15)6 = 0.007. Hence, the three-recombinant consensus region is akin to a 99% credible interval. The same probabilities for the one- and two-recombinant regions are 0.28 and 0.04, respectively. Of course, this credible interval is clearly influenced by the ‘sporadic’ rate considered. It should be noted, however, that meaning of sporadic in this situation is the probability that a confirmed PRCA case who shares a segregating haplotype in a linked pedigree with a least 3 other confirmed PRCA cases, is, in fact, merely a phenocopy.
We would like to express our gratitude to the many families who participated in the many studies involved in the International Consortium for Prostate Cancer Genetics (ICPCG). The ICPCG, including the consortium’s Data Coordinating Center (DCC), is made possible by a grant from the National Institutes of Health U01 CA89600 (to W.B.I.). N.J.C. was supported in part by USPHS CA98364 (to N.J.C.). Additional support to participating groups, or members within groups, is as follows:
AAHPC Group: The authors would like to express their gratitude to the African-American Hereditary Prostate Cancer Study (AAHPC) families and study participants for their continued involvement in this research. We specifically name, C. Ahaghotu, J. Bennett, W. Boykin, G. Hoke, T. Mason, C. Pettaway, S. Vijayakumar, S. Weinrich, M. Franklin, P. Roberson, J. Frost, E. Johnson, L. Faison-Smith, C. Meegan, M. Johnson, L. Kososki, C. Jones and R. Mejia. We would also like to thank members of the National Human Genome Center (NHGC) at Howard University namely R. Kittles, G.M. Dunston, P. Furbert-Harris and C. Royal. We would also like to acknowledge the contribution of the National Human Genome Research Institute (NHGRI) and TGen genotyping staff including E. Gillanders and C. Robbins. The AAHPC study would not have been possible without F. Collins (Director of NHGRI) and J. Trent (Director of TGen). This research was funded primarily through the NIH Center for Minority and Health Disparities (1-HG-75418). A.B.B-B and A.G also received support from USPHS CA-06927 and an appropriation from the Commonwealth of Pennsylvania. This research was also supported in part by the Intramural Research Program of the NIH (NHGRI) and USPHS RR03048 from the National Center for Research Institute and USPHS RR03048 from the National Center for Research Resources.
ACTANE Group: Genotyping and statistical analysis for this study, and recruitment of U.K. families, was supported by Cancer Research U.K (CR-UK). Additional support was provided by The Prostate Cancer Research Foundation, The Times Christmas Appeal and the Institute of Cancer Research. Genotyping was conducted in the ‘Jean Rook Gene Cloning Laboratory’ which is supported by BREAKTHROUGH Breast Cancer - Charity No. 328323. The funds for the ABI 377 used in this study were generously provided by the legacy of the late Marion Silcock. We thank S. Seal and A. Hall for kindly storing and logging the samples that were provided. D.F.E is a Principal Research Fellow of CR-UK. Funding in Australia was obtained from The Cancer Council Victoria, The National Health and Medical Research Council (grants 940934, 251533, 209057, 126402, 396407), Tattersall’s and The Whitten Foundation. We would like to acknowledge the work of the study coordinator M. Staples and the Research Team B. McCudden, J. Connal, R. Thorowgood, C. Costa, M. Kevan, and S. Palmer, and to J. Karpowicz for DNA extractions. The Texas study of familial prostate cancer was initiated by the Department of Epidemiology, M.D. Anderson Cancer Center. M.B. was supported by an NCI Post-doctoral Fellowship in Cancer Prevention (R25). We would also like to specifically thank the following members of ACTANE: S. Edwards, M. Guy, Q. Hope, S. Bullock, S. Bryant, S. Mulholland, S. Jugurnauth, N. Garcia, A. Ardern-Jones, A. Hall, L. O’Brien, B. Gehr-Swain, R. Wilkinson, D. Dearnaley, The UKGPCS Collaborators, British Association of Urological Surgeons’ Section of Oncology (UK Sutton); Chris Evans (UK Cambridge); M. Southey (Australia); N. Hamel, S. Narod, J. Simard (Canada); C. Amos (USA Texas); N. Wessel, T. Andersen (Norway); D.T. Bishop (EU Biomed). BC/CA/HI Group: USPHS CA67044. FHCRC Group: USPHS CA80122 (to J.L.S.) which supports the family collection; USPHS CA78836 (to E.A.O). E.A.O was supported in part by the NHGRI. JHU Group: Genotyping for the JHU, University of Michigan, University of Tampere, and University of Umeå groups’ pedigrees was provided by NHGRI and TGen genotyping staff including E. Gillanders, MP Jones, D. Gildea, E. Riedesel, J. Albertus, D. Freas-Lutz, C. Markey, J. Carpten and J. Trent. Mayo Clinic Group: USPHS CA72818. Michigan Group: USPHS CA079596. University of Tampere Group: The Competitive Research Funding of the Pirkanmaa Hospital District, Reino Lahtikari Foundation, Finnish Cancer Organisations, Sigrid Juselius Foundation, and Academy of Finland grant 211123. University of Ulm Group: Deutsche Krebshilfe, grant number 70-3111-V03. University of Umea Group: Work was supported by the Swedish Cancer Society and a Spear grant from the Umeå University Hospital, Umeå, Sweden. University of Utah Group: Data collection was supported by USPHS CA90752 (to L.A.C.-A.) and by the Utah Cancer Registry, which is funded by Contract #N01-PC-35141 from the National Cancer Institute’s Surveillance, Epidemiology, and End-Results Program with additional support from the Utah State Department of Heath and the University of Utah. Partial support for all datasets within the Utah Population Database was provided by the University of Utah Huntsman Cancer Institute and also by the USPHS M01-RR00064 from the National Center for Research Resources. Genotyping services were provided by the Center for Inherited Disease Research (N01-HG-65403). DCC: The study is partially supported by USPHS CA106523 (to J.X.), USPHS CA95052 (to J.X.), and Department of Defense grant PC051264 (to J.X.).