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IV. APPLICATIONS PANEL Confirmation of Prostate Cancer Susceptibility Genes Using High-Risk Families Gail P. Jarvik, Janet L. Stanford, Ellen L. Goode, Richard McIndoe, Suzanne Kolb, Mark Gibbs, Leroy Hood, Elaine A. Ostrander Prostate cancer is the most common nonsquamous cell cancer and the second leading cause of cancer deaths among men in the United States. In 1998, approximately 184 500 men in the United States will be clinically diagnosed with prostate cancer and, despite advances in treatment and screening, an estimated 39 000 men will die of the disease (1). Incidence rates increased sharply from 1973 to 1992 (particularly among white men), with the increase being attributed to screening and early detection of tumors through the use of testing for prostate-specific antigen (PSA) screening (2–4). Little is known about the underlying causes of prostate cancer. A number of environmental risk factors have been considered, including industrial exposures, sexually transmitted infectious agents, endocrine factors, and diet. High dietary fat may be associated with invasive prostate cancer (5–9). Evidence also suggests that vitamins D (10–12) and E (13) may have protective effects. A cause–effect relationship has yet to be demonstrated in either of these cases, however, and no particular environmental factor has been identified as a major cause of the disease. One of the strongest risk factors for prostate cancer identified to date is a family history of disease, suggesting that genetic factors play a role in prostate cancer susceptibility, etiology, and progression. We discuss here the key factors that suggest a role for prostate cancer susceptibility genes and the likely features of such genes. We review findings that suggest that there is a prostate cancer susceptibility gene at 1q24–25. Finally, we discuss the results in our dataset and others with regard to observed difficulties in confirming these initial findings of linkage. EPIDEMIOLOGIC ANALYSES A number of epidemiologic studies demonstrate familial clustering of prostate cancer, suggesting the existence of underlying Journal of the National Cancer Institute Monographs No. 26, 1999 genetic predisposition factors. A subset of those studies is summarized in Table 1 (14–21). In each study, family history was evaluated as a risk factor for prostate cancer. When defining exposure as a positive first-degree family history of prostate cancer (father, brother, or sons), the frequency of exposure is about threefold higher among case subjects than among control subjects, with relative risk (RR) estimates across studies ranging from 2.0 to 6.3. Two studies (22,23) have suggested that elevated risk may be associated with affected brothers but not with affected fathers, suggesting a possible X chromosome or shared childhood environment risk. Overall, men with two or more first-degree-affected relatives have a fivefold to 11-fold increase in risk compared with men without a family history of prostate cancer (16,24,25). These studies also provide evidence that the risk of prostate cancer in first-degree relatives of probands is higher among patients with early, compared with late, ages at diagnosis. A genetic contribution to prostate cancer risk is also supported by twin studies. In one such analysis, Grönberg et al. (26) analyzed the incidence of disease among 3840 male twin pairs identified from the Swedish Twin Registry linked to the Swedish Cancer Registry. The study population contained 458 cases of prostate cancer. Results suggest a significant influence of genetics on prostate cancer with proband concordance rates of 0.19 for monozygotic twins and 0.043 for dizygotic twins. Subsequent studies (27,28) in these and additional cohorts confirmed these initial observations. MOLECULAR ANALYSIS A number of loci on different chromosomes have been implicated in the etiology of prostate cancer. These results are not mutually exclusive and suggest that multiple genes contribute to the progression of prostate tumors and that some of these genes may have redundant functions. Studies on allelic loss in sporadic prostate tumors have been a common approach for identifying regions likely to contain prostate cancer genes [e.g. (29–31)]. Affiliations of authors: G. P. Jarvik (Department of Medicine, Division of Medical Genetics), E. L. Goode (Department of Epidemiology, School of Public Health and Community Medicine), R. McIndoe, L. Hood (Department of Molecular Biotechnology), University of Washington Medical Center, Seattle; J. L. Stanford, S. Kolb (Division of Public Health Sciences), M. Gibbs, E. A. Ostrander (Clinical Research Division), Fred Hutchinson Cancer Research Center, Seattle. Correspondence to: Elaine A. Ostrander, Ph.D., Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave., N., D2-190, Seattle, WA 98109-1024 (e-mail: [email protected]). See “Notes” following “References.” © Oxford University Press 81 Downloaded from http://jncimono.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014 Data from many types of studies support the hypothesis that strong familial components are involved in the etiology of prostate cancer. One way to access such genes is through the study of families with multiple affected family members and, in particular, families with individuals affected comparatively early in life. Several prostate cancer susceptibility loci have been described to date. Confirmation of the linkage and estimation of the proportion of families who are linked in large independent datasets is essential to understanding the significance of susceptibility genes. We explore the methodology used to perform such studies and the factors that can limit the ability to confirm linkage results. We report specifically the example of the HPC1 gene on 1q24–25. [Monogr Natl Cancer Inst 1999;26:81–7] Table 1. Epidemiologic studies of prostate cancer in relation to family history of prostate cancer No. of subjects Study, y (reference No.) Krain, 1974 (14) Fincham et al., 1990 (15) Steinberg et al., 1990 (16) Spitz et al., 1991 (17) Hayes et al., 1995 (18) Whittemore et al., 1995 (19) Lesko et al., 1996 (20) Ghadirian et al., 1991 (21) Positive family history (%) Estimated relative risk* Case subjects Control subjects Case subjects Control subjects (95% CI or P) 221 382 691 378 972 1500 563 640 221 625 640 383 1294 1581 703 639 6.0 15.0 15.0 13.0 6.9 13.0 17.2 14.7 1.0 5.0 8.0 5.7 2.3 5.0 8.3 5.0 6.3 ( P<.01) 3.4 (2.2–5.3) 2.0 (1.2–3.3) 2.4 (1.3–4.5) 3.2 (2.0–5.0) 2.5 (1.9–3.3) 2.2 (1.5–3.0) 3.2 (2.1–5.1) *CI ⳱ confidence interval. COMPLEX SEGREGATION ANALYSIS Complex segregation analysis (CSA) is useful for testing the hypothesis that a mendelian locus substantially contributes to the 82 risk of a disease. CSA can give information about the likely mode of inheritance and penetrance of prostate cancer susceptibility genes. It can also suggest subsets of families, or the characteristics of men within families, whose disease is most likely because of mutations in an inherited susceptibility gene. CSA cannot, however, reliably distinguish the number of susceptibility loci nor can it provide information about the location of susceptibility genes. CSA can provide estimates of the overall frequency of mutant alleles with a specific set of characteristics, the penetrance of these alleles, and other parameters of the transmission models that can be used in linkage analyses. Several major CSAs of prostate cancer have been undertaken. The first, undertaken by Carter et al. (24) analyzed data on 691 prostate cancer families, originally ascertained from consecutive probands (mean age, 59.3 years) undergoing radical prostatectomy for primary clinically localized prostate cancer at The Johns Hopkins University Hospital, Baltimore (MD) between 1982 and 1989. These data showed that early age of diagnosis and multiple affected family members were the strongest predictors of risk in relatives. Their analysis also suggested that autosomal dominant inheritance of a rare, highly penetrant allele(s) best accounted for the distribution of prostate cancer in the families studied, with carriers having an 88% cumulative risk of prostate cancer by age 85 years compared with only 5% cumulative risk for noncarriers. These investigators proposed that hereditary prostate cancer gene(s) account for about 43% of prostate cancer with diagnosis before age 55 years and about 9% of total prostate cancer. Similar results have been obtained by Schaid et al. (43) with the use of a family-history survey of cancer conducted for 5486 men, including 4288 probands (aged 38–84 years) who underwent a radical prostatectomy for clinically localized prostate cancer at the Mayo Clinic between 1966 and 1987. This study proposed a population frequency of 0.006 for a susceptibility gene and a risk of 89% by age 85 years for carriers. This study, however, suggested that autosomal dominant genes accounted for a higher proportion of all prostate cancer—approximately 68% before age 60 years. An additional segregation analysis was performed on a population-based sample of 2857 nuclear families ascertained through an affected father diagnosed with prostate cancer in Sweden between 1959 and 1963 (44). This analysis contrasts with the studies by Carter et al. (24) and Schaid et al. (43), who both studied hospital-based series of men whose disease was treated with prostatectomy. Selection of probands by prostatectomy may bias selection toward men with less aggressive disJournal of the National Cancer Institute Monographs No. 26, 1999 Downloaded from http://jncimono.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014 Thus far, loss of heterozygosity (LOH) in prostate tumors has been documented for at least 11 different chromosomal arms. Many of these regions have been identified repeatedly, such as chromosomes 8 and 17 (32,33). Chromosome 17 is of interest because it carries a number of genes postulated to have a role in cancer, including the p53 and BRCA1 genes. Of interest, families that carry BRCA1 mutations have been reported to be at increased risk for prostate cancer (RR 3.33; 95% confidence interval ⳱ 1.8–6.2) (34). Analyses of prostate cancer cases drawn from a population-based, case–control study, however, revealed little evidence that BRCA1 is a common prostate cancer susceptibility gene (35). In 49 prostate cancer cases who had either been diagnosed early in life (<53 years), were diagnosed before age 65 years, and had first-degree family history of breast cancer or an early onset family history of prostate cancer, only one germline BRCA1 mutation was observed. There is a likely possibility that this single observation was coincidental, as was the delAG1285 mutation that has been frequently observed in the population. This finding suggests that LOH observed on chromosome 17q in many prostate tumors is likely to be unrelated to the presence of germline mutations in BRCA1. Other techniques that highlight regions of interest include chromosome transfer studies and comparative genome hybridization that implicate chromosomes 2, 12, 11, and 8 (36–39). The data from chromosome 8 are particularly interesting because LOH studies also implicate chromosome 8. However, exhaustive linkage analysis of chromosome 8 by a variety of methods has yet to reveal a gene important in prostate cancer predisposition (40–41). The identity, penetrance, and role in cancer susceptibility of each of the above postulated genes remain unknown. Two points are clear, however. First, whereas the location and number of the underlying prostate cancer susceptibility genes remain elusive, collectively these studies argue that tumor suppression is a key function of at least some prostate cancer genes [reviewed in (42)]. Second, many of these genes are likely to be more important in prostate cancer progression and metastasis and do not represent early events in the development of prostate cancer. For the mapping of susceptibility genes, discussed below, analyses of families with a high incidence of prostate cancer are needed. ease, since metastatic disease is unlikely to be treated by prostatectomy. Grönberg et al. (44) reported that familial clustering of prostate cancer was best explained by dominant alleles with a population frequency of 0.0167 and a lifetime penetrance of 63%, slightly less than the penetrance proposed by Carter et al. Nevertheless, the overall consistency in segregation analyses derived from very different populations provide strong evidence for highly penetrant autosomal dominant prostate cancer susceptibility genes. PUTATIVE PROSTATE CANCER LOCUS AT 1Q24–25 Journal of the National Cancer Institute Monographs No. 26, 1999 83 Downloaded from http://jncimono.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014 In 1996, the location of a putative prostate cancer susceptibility locus (termed HPC1) on chromosome 1q24–25 was suggested by linkage analysis of 91 high-risk prostate cancer families from the United States and Sweden (40). A maximum multipoint logarithm of the odds (LOD) score under the assumption of heterogeneity LOD (HLOD) ⳱ 5.43 (the proportion of families linked, ␣ ⳱ 0.34), with the postulated locus being close to marker D1S422. This study concluded that about 34% of inherited prostate cancer could be attributed to an as yet unknown gene at 1q24–25. The above conclusions were strongly supported by a nonparametric analysis using the program GENEHUNTER (45) in which highly significant results were obtained for multiple markers in this region. Although the initial report did not suggest any subgroup of families that were more or less likely to be linked, a strong age of diagnosis effect, with nearly all linked families having an average age of diagnosis of less than 65 years has since been noted. Specifically, Grönberg et al. (46) found that the 40 North American families with an average age of diagnosis less than 65 years yielded a multipoint LOD score of 3.96, whereas the 39 families with an older age at diagnosis yielded a LOD score of −0.84. Assuming heterogeneity, the proportion of families linked was estimated as 66% for the 14 families with the earliest average ages at diagnosis but decreased to 7% for the families with the oldest ages at diagnosis. The majority of families contributing to the positive LOD score in each case were Caucasian. Grönberg et al. (46,47) also reported that high-grade cancers (grade 3) were more common in potentially linked families as was advanced-stage disease. These latter two points have been the topic of some dispute, however, with claims that the comparison of characteristics between putatively linked and unlinked families is faulty because of the use of an incorrect reference population (48). It should be noted that conclusions regarding clinical characteristics of linked families are virtually impossible to make with any certainty until the HPC1 gene is cloned. The presence of a modestly positive LOD score, or even completely shared haplotypes from the chromosomal region in question among affected individuals, does not guarantee that the family is “linked,” since such segregation will occur fortuitously some percentage of the time. Conversely, there will be families with low LOD scores but who have a mutation in the gene in question. The LOD scores may be low because of phenocopies, i.e., men who have acquired the same disease for “sporadic” reasons unrelated to germline mutations for the gene in question. In addition, because of variable penetrance of mutations, there will be families in which carriers of the mutation develop the disease at a point later in life then predicted by the statistical models or not at all. Therefore, conclusions regarding clinical characteris- tics of “linked families” will be best drawn from analyses of families who are found to carry true germline mutations, following characterization of the causative gene. Shortly after the mapping of HPC1, we described an analysis (49) of 49 high-risk prostate cancer families for linkage to 1q24– 25 with the use of 10 highly polymorphic markers spanning the proposed locus. These 49 families were representative of our families as a whole, which were collected from the Prostate Cancer Genetic Research Study (PROGRESS), a Seattle-based study initiated in July 1995 to identify familial clusters of prostate cancer and, ultimately, inherited susceptibility genes. A tollfree number (1-800-777-3035) was established and distributed via national advertising, media events, and mailings to support groups and urologists to identify potential participants for their suitability. Informed, written consent was obtained from each study participant. The study and its consent and medical record release forms were approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center. Families were selected for participation on the basis of the number of firstdegree relatives diagnosed with prostate cancer, the age at diagnosis of the affected relatives, and the number of living affected relatives from whom blood samples could be obtained. In our first set of 49 families, we observed that the characteristics of the families analyzed compared closely with those described by Smith et al. (40). These PROGRESS families averaged 4.4 men affected per family (range, 3–9) and had an average age at diagnosis of 65.9 years. Forty-six of the 49 PROGRESS families were Caucasian. Data were analyzed by use of parametric and nonparametric methods. Two models were used for the parametric LOD score method. The first model (“Hopkins model” or “Model A”) was identical to that reported by Smith et al. (40), and the second (“Seattle model” or “Model S1”) (49) was devised by us and based on the age-dependent penetrance values that resulted from the segregation analysis reported by Carter et al. (24). In addition, to protect against falsely rejecting linkage secondary to model misspecification, we also performed a nonparametric multipoint analysis with the use of the GENEHUNTER program (45). In all cases, we observed no evidence for linkage in our dataset in either two-point analyses (using any of 10 markers in the region) or in the nonparametric analyses. No evidence for linkage was observed in a heterogeneity analysis using HOMOG. More recently, we have expanded our dataset to include 150 families (Table 2; 144 Caucasian and six non-Caucasian) and undertaken a closer examination at this locus with the use of three linkage models: the Seattle model (49), the model described by Smith et al. (40), and a model used in a recent linkage analysis published by Grönberg et al. (46). These complete results (50), including features of the families analyzed, are similarly negative with no evidence for linkage with a subset of families at 1q24–25. Table 3 presents the results from the 150 multiplex prostate cancer families described in Table 2 for one marker, D1S518, which defines the HPC1 locus. The results are highly negative with all models and all values of , as well for all other markers analyzed (50). One consideration in this analysis is the degree to which the families analyzed mimic those described by Smith et al. (40). The mean age of diagnosis of the genotyped affected men is 65.9 years, and the average number of affected men per family is 4.2 (Table 3). These figures are comparable to that of Smith et al. Table 2. Characteristics of 150 prostate cancer families from the PROGRESS study* Caucasian Characteristic Total group Non-Caucasian 艋65 y 艋65 y >65 y >65 y No. of affected men 637 275 328 26 8 No. of genotyped affected men 509 219 263 20 7 Average No. of affected men per family (range) Mean age at diagnosis of genotyped affected men per family (range) 4.2 (3–9) 4.3 (3–9) 4.1 (3–8) 6.5 (4–9) 4.0 (3–5) 65.9 (50.8–78.0) 62.0 (50.8–66.0)† 69.5 (65.0–78.0) 62.2 (57.7–63.7) 69.3 (67.0–71.7) *PROGRESS ⳱ Prostate Cancer Genetic Research Study. †Men genotyped at 1q24 are not identical to the overall set of affected men samples. Table 3. LOD* scores for 150 prostate cancer families using three transmission models at 1q for marker D1S518 S1 (49) A (40) B (46) 0.00 0.02 0.04 0.08 0.10 0.20 0.30 0.40 −30.98 −84.51 −12.46 −24.28 −50.94 −10.25 −19.41 −37.47 −8.51 −12.66 −22.72 −5.92 −10.24 −18.04 −4.93 −3.25 −5.65 −1.84 −0.74 −1.38 −0.54 −0.08 −0.17 −0.09 *LOD ⳱ logarithm of the odds. (40) in the description of families collected by investigators at The Johns Hopkins University. LOCUS HETEROGENEITY LINKAGE AND POWER TO DETECT The inability to robustly replicate the findings of Smith et al. (40) suggests that locus heterogeneity issues for HPC1 are still not well understood. There may be diagnostic differences between datasets or it may be that a prostate cancer gene at 1q24– 25 accounts for significantly less than 34% of disease in “highrisk” families nationwide. How much less is unclear. In our dataset, we have undertaken an analysis to estimate the power to detect linkage. Conservative computations suggest a high power to detect a hereditary prostate cancer (HPC) locus (or loci) by use of a multistep analysis strategy in this large dataset. Power calculations for two-point (one marker and HPC) LOD method analyses were done with 1000 replications with the use of the computer package SIMLINK (51,52). The simulations computed the probability of detecting certain LOD scores, given the autosomal dominant model of HPC transmission discussed above, the minimum pedigree structures, and the DNA samples available. The computations presented are conservative in sev- eral ways. They used only the pedigree structure that was available in the time of initial ascertainment. Much more data have been collected on many families in the intervening months. The power analysis also assumed a polymorphism information content (PIC) of 0.7 for the marker loci. A marker with a PIC of 0.8, as several of the markers in the HPC region have, would confer additional power. Another consideration is that, in the case of HPC1, we are conducting a confirmation study with dense markers and, therefore, expect to be small. Table 4 calculates power for ⳱ 0.00 and 0.05. For the HPC1 region, the power analysis for ⳱ 0.00 is reasonable. Finally, at the time power analyses were completed, only 114 families were collected, half of which had a mean age of familial prostate cancer less than 65 years and half of which had a mean age of 65 years or greater. The most recent molecular analyses were done with 150 families, approximately half of whom have a mean age under 65 years, using the age-dependent penetrance model (Seattle model) we have described previously. Despite these caveats, power analysis of 114 families indicates acceptable power to detect linkage, even if a locus accounted for less than 35% of prostate cancer in the dataset. Application of these findings argues that, at least in this dataset, significantly less than 35% of families are linked to 1q24–25. Table 4. Power analysis of 114 Caucasian families with mean age of diagnosis <65 years* % linked <65 y % linked total dataset 100 50 90 45 80 40 70 35 Expected LOD Probability LOD > 1 Probability LOD > 1.5 Probability LOD > 2 Probability LOD > 3 0 0.05 0 0.05 0 0.05 0 0.05 11.448 7.264 7.264 4.751 4.581 3.132 2.741 1.915 1.000 0.999 0.999 0.988 0.988 0.929 0.908 0.749 1.000 0.996 0.996 0.956 0.952 0.832 0.744 0.542 1.000 0.993 0.993 0.905 0.890 0.684 0.590 0.353 1.000 0.975 0.975 0.816 0.789 0.495 0.399 0.202 *LOD ⳱ logarithm of the odds. 84 Journal of the National Cancer Institute Monographs No. 26, 1999 Downloaded from http://jncimono.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014 Recombination fraction Model (reference No.) COMPARATIVE ANALYSES EFFECTS OF HETEROGENEITY There is currently linkage evidence for four familial prostate cancer loci (40,57,59–61). Because these loci do not account for more than one half of the families reported, other loci are likely to exist. Locus heterogeneity dramatically influences the power to map. This can be seen by examining the fall in expected LOD score as heterogeneity increases. Table 5 demonstrates the tradeoff between sample size, heterogeneity, and power in the simulation studies for our late-diagnosis families. Total sample sizes of 116 and half that, 58 families, are examined. It can be seen that increasing the proportion of families linked to the locus of interest from 60% to 70% or from 70% to 80% results in a nearly Table 5. Caucasian families with mean age of diagnosis for genotyped affected men >65 years* No. of families Linked, % Max LOD Probability LOD > 1 Probability LOD > 2 Probability LOD > 3 116 90 116 80 116 70 116 60 58 100 58 90 58 80 58 70 0 0.05 0 0.05 0 0.05 0 0.05 0 0.05 0 0.05 0 0.05 0 0.05 9.362 6.124 5.915 4.030 3.585 2.441 2.064 1.443 7.071 4.608 4.608 3.127 3.100 2.111 1.833 1.294 1.000 1.000 1.000 0.981 0.971 0.876 0.796 0.643 0.999 0.992 0.992 0.922 0.912 0.754 0.663 0.510 0.999 0.977 0.976 0.850 0.762 0.515 0.402 0.220 0.995 0.932 0.932 0.761 0.730 0.472 0.413 0.240 0.998 0.931 0.923 0.699 0.578 0.290 0.212 0.081 0.972 0.770 0.770 0.473 0.451 0.221 0.161 0.077 *LOD ⳱ logarithm of the odds. Journal of the National Cancer Institute Monographs No. 26, 1999 CONFOUNDING FACTORS We believe that the two factors that most likely complicate locus heterogeneity issues in the mapping of prostate cancer genes are PSA testing and race. PSA came into common usage as a diagnostic tool in 1988. Because different datasets have collected men spanning different diagnosis years, the proportion of men with less aggressive disease who were diagnosed by PSA and categorized simply as “affected” likely differs (perhaps significantly) between datasets and within individual strata. A weighting factor based on the level of PSA in the linkage analyses may shed light on this issue, as may stratification by tumor stage and grade. Whether or not race is an issue for HPC1 analysis remains unclear. Only two families in the Seattle dataset were AfricanAmerican. It is clear that prostate cancer is more frequent among U.S. blacks than whites (62) and that it is lowest in Asian men. Although the majority of families contributing to the HPC1 linkage results are Caucasian in all datasets, in the work described by Cooney (53), several African-American families contributed to the observation of linkage, with a maximum NPL Z score at marker D1S158 of 1.39 (P ⳱ .0848). There was also an overrepresentation of African-American families contributing to the positive LOD score in the original report of Smith et al. (40) as well, generating the hypothesis that the gene at 1q24–25 may be more important for prostate cancer susceptibility in AfricanAmerican than in Caucasian families. In part, because the results in individual families are not strong enough to conclude linkage of that family to the 1q24–25 region, our understanding of any differential effect of the HPC1 locus awaits larger collections of non-Caucasian families. CONCLUSIONS The confirmation of the HPC1 locus has not been uneventful. The mixed results found by several groups who have tried to confirm this linkage are likely the result of population differences in the samples and a lower proportion of families being linked than was originally estimated. The mixed results do not necessarily indicate that this linkage is a false positive, rather that the HPC1 gene may be differentially important for families with different epidemiologic or clinical characteristics. It remains for the HPC1 gene to be cloned and sequenced before most families whose disease is positively because of mutations in HPC1 can be unambiguously identified. 85 Downloaded from http://jncimono.oxfordjournals.org/ at Pennsylvania State University on February 27, 2014 The strong HPC1 linkage observed by Smith et al. (40) has proven challenging to replicate in other datasets, with mixed results generated by seemingly similar sets of families. By applying nonparametric analyses with the use of GENEHUNTER to studies of families with significant numbers of affected young men, data from Cooney et al. (53) and Hsieh et al. (54) weakly confirm the observations of Smith et al., with nonparametric linkage scores of 1.72 and 1.83, respectively, and P values of .045 and .036. The significance of NPL scores with P values in the .03–.05 range for a confirmation result is debatable. In a detailed discussion on this topic, Lander and Kruglyak (55) have suggested that a nominal P value of .01 should be required for confirmatory evidence of linkage, since such studies still involve searching over an interval. That P values <.05 were seen in two distinct datasets, however, strengthen the argument for confirmation in these datasets. The percentage of linked families in the overall datasets cannot be estimated from these analyses but will be much less than 34%. In contrast to the above, Eeles et al. (56) and Berthon et al. (57) found no evidence for linkage at this locus. Eeles et al. reported an NPL score of 0.72 with a P value of .22 in 35 families with four affected men. In these same families, the HLOD ⳱ 0.20, with ␣ ⳱ 20%. The HLOD for the larger dataset of 136 families was 0.01, ␣ ⳱ 4%, suggesting that only 4% of the families were linked to the HPC1 locus. Virtually identical negative data have been presented by Thibodeau et al. (58). 50% drop in the sample size for required equivalent power. This demonstrates the advantage obtained if homogeneous subsets can be constructed for linkage testing. For prostate cancer, families have been divided by age of diagnosis, ethnicity, and density or severity of disease. Homogeneous subsets may also be improved by stratifying on the LOD score at other putative loci. Alternately, linkage methods in which two or more loci may be jointly considered may be advantageous when locus heterogeneity is suspected within a sample. The problem of heterogeneity also sheds light on how different samples, ascertained in different ways, may enrich their collection for certain clinical features and, therefore, may have different mixes of susceptibility loci and would result in the differential ability to map each locus between samples. 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