Psychiatry Elsevier
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Failure to Replicate Linkage Between Chromosome 5qll -ql3 Markers and Schizophrenia in 28 Families Dominique Campion, Thierry d’Amato, Hassan Laklou, Olivier Sabate, Maurice Jay, Marion Leboyer, Alain Malafosse, Philip Gorwood, Marie Claude Babron, Dominique Hillaire, Franqoise Clerget-Darpoux, Gilles Waksman, and Jacques Mallet Received January 7, 1992; revised version received May 26, 1992; accepted September 5, 1992.
Abstract. Sherrington et al. (1988) reported linkage between markers located on the 5ql l-q13 region of chromosome 5 and schizophrenia in five Icelandic and two British families. To date, however, all attempts to replicate the initial finding have failed. Using three markers of chromosome 5, we have studied 28 additional French pedigrees. When our data were analyzed both with parametric (i.e., lod scores) and nonparametric methods, we found no evidence of linkage. Thus, we were unable to replicate the earlier report by Sherrington et al. Key Words. Genetic analysis, molecular genetics, affected-pedigree member method.
Despite significant evidence from adoption and twin studies that genetic factors play a role in the etiology of schizophrenia (Gottesman and Shields, 1985), the mode of inheritance of the disease remains elusive (McGue et al., 1985). Segregation analyses have yielded ambiguous results. Basically, the methodology of such studies suffers from intrinsic limitations-for example, the oversimplification of the models used and the assumption of genetic homogeneity (Baron, 1986). Advances in tiolecular genetics offer the opportunity to develop an alternative strategy for the detection of genetic risk factors. Indeed, the linkage approach, which studies the cosegregation between DNA markers and diseases within pedigrees, is a powerful tool to characterize susceptibility genes. This strategy has already achieved success in Mendelian diseases such as cystic fibrosis (Riordan et al., 1989) or Huntington’s chorea (Gusella et al., 1983). With the report by Sherrington et al. (1988) of linkage between schizophrenia and genetic markers located on the long arm of chromosome 5, it seemed that a gene might have been identified that predisposes to a genetically complex disease. Unfortunately, subsequent investigators have failed to detect linkage with chromosome
Dominique Campion, M.D., Marion Leboyer, M.D., Ph.D., Philip Gorwood, M.D., Marie Claude Babron, Ph.D., and FranCoise Clerget-Darpoux, Ph.D., are at the “Unit& de Recherche d%pidtmiologie G&nttique”(INSERM U 155), Chateaude Longchamp, 75016 Paris. Maurice Jay, M.D., is at Hbpital St Paul, La Reunion. Dominique Hillaire, Ph.D., is at “Genethon,” Evry. Thierry d’Amato, M.D., Hassan Laklou, Research Assistant, Olivier Sabate, M.D., Alain Malafosse, M.D., Gilles Waksman, Ph.D., and Jacques Mallet, Ph.D., are at “Laboratoire de Neurobiologie MoKzculaire et Cellulaire”(CNRS), 91190 Gif_sur Yvette. (Reprint requests to Dr. D. Campion, Centre Hospitalier Specialis& rue P. Eluard, Bolte Postale 45, 76301 Sotteville-l&s-Rouen, France.) 0165-1781/92/$05.00
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172 5ql l-q13 markers (Kennedy et al., 1988; Detera-Wadleigh et al., 1989; Kaufmann et al., 1989; St. Clair et al., 1989; Aschauer et al., 1990; McGuffin et al., 1990; Crowe et al., 1991). It could be argued that some of these studies (Kennedy et al., 1988; Detera-Wadleigh et al., 1989; St. Clair et al., 1989) are not true “replications” of the study of Sherrington et al. (1988) since the diagnostic procedures and the genetic parameters of the original report were not used. However, the models used in the various studies are similar enough to suggest that differences in parameter values cannot account for such discordant results. The contention that a susceptibility locus for schizophrenia can be excluded from chromosome 5qll-q13 and that the positive results of Sherrington et al. occurred by chance (McGuffin et al., 1990) is controversial (Amos et al., 1991). As the use of the lod score’ method in genetically complex diseases raises many methodological questions (Merikangas et al., 1989; Baron et al., 1990), it seemed appropriate to conduct a new replication study that combined the lod score analysis and a nonparametric approach (i.e., the affected pedigree member method; Weeks and Lange, 1988). To conduct this replication study, we therefore collected new multiplex pedigrees but found no evidence of linkage between schizophrenia and chromosome 5 markers. Methods Psychiatric admissions were systematically Family Ascertainment and Diagnoses. screened in two locations: the city of Rouen (France) and the island of la Rtunion (Indian Ocean). Details about ascertainment and diagnostic procedures are given elsewhere (Leboyer et al., 1990). In brief, families were ascertained on the basis of possessing at least two first degree relatives with a diagnosis of schizophrenia. Pedigrees showing evidence of assortative mating between schizophrenic subjects were discarded. Paternity was confirmed by human leukocyte antigen and red cell typing. Two semistructured interviews were used in the clinical assessment of probands and available relatives: the French translations of the Schedule for Affective Disorders and Schizophrenia-Lifetime Version (SADS-LA; Fyer et al., 1985) and the Schedule for Schizotypal Personalities (SSP; Baron et al., 1981). In addition, hospital charts were consulted. Best-estimate diagnoses according to DSM-III criteria (American Psychiatric Association, 1980) were made by two raters without knowledge of pedigree structure. Marker status was determined without knowledge of clinical diagnosis. Twenty-eight families (14 in metropolis and 14 in la Reunion) were included in this study. In these 28 families, there were 91 cases of schizophrenia, 3 cases of schizoaffective disorders, and 11 cases of schizophrenia spectrum disorders (schizotypal and paranoid personalities). Five individuals were classified as “unknown phenotype” and 21 as “other psychiatric disorders” (including 8 diagnosed as suffering from alcohol abuse, 4 from major depression, 3 from phobic disorder, 1 from obsessive-compulsive disorder, 3 from mental retardation (mild), 1 from dysthymic disorder, and 1 from somatization disorder). No individuals afflicted with bipolar disorder were found in our sample. Pedigrees are available from the authors on request. Restriction Fragment Length Polymorphism Typing. Venous blood samples were obtained from subjects and cell lines were estabished by Epstein-Barr virus transformation of fl lymphocytes (Neitzel et al., 1986). High molecular weight DNA was extracted from cultured I. The lod score = the log to the base IO of the probability that a given set of data about genetic recombination would arise by virtue of two loci being linked at a specified recombination fraction divided by the probability that the data would arise by nonlinkage.
173 cells, digested with Msp 1 and Taq 1 restriction enzymes, electrophoretically separated on agarose gel and transferred to nylon membranes by standard methods (Maniatis et al., 1982). The filters were hybridized with J2P radiolabeled probes. Locus D5 S76 was defined by a Taq 1 polymorphism detected by probe p105-599ha, and loci D5 S39 and D5 S78 were defined by Msp 1 polymorphisms detected by probes p105-153ra and p105-798rb, respectively. Table 1 provides details about the probes used, and the length and frequencies of the fragments recognized. On the basis of analysis of the families from the Centre d’fitude du Polymorphisms Humain (Weiffenbach et al., 1991), the order and approximate recombination distances between the markers are: p105-599ha (19 CM) p105-153rb (12 CM) p105-798ha.
Table 1. Chromosome 5 markers Marker 105~599ha
105-153ra 105-79&b
(locus)
Location
Polymorphism
5q12-14
Taq 1
5q12-14 5q12-14
Mspl Mspl
Alleles (kb)
Frequencies
al = 17.00
0.32.
a2 = 14.00
0.16
a3 = 10.00
0.50
a4 =
0.02
9.00
bl = 8.70
0.33
b2 = 5.80
0.67
cl = 2.30
0.57
c2=
0.43
1.80
Linkage Analysis. Two-point linkage analyses were performed with MLINK (Lathrop et al., 1985) using the same clinical classifications and genetic parameters as Sherrington et al. (1988) To define the pathological phenotype, we first used the clinical classification that gave the maximum lod score in the Sherrington et al. report. In this classification, called DOMSSF, all subjects with a psychiatric diagnosis were scored as affected. However, since this categorization is very unusual and since the more conventional DOMSS classification (which includes individuals with schizophrenia and schizophrenia spectrum disorders) also gave a positive lad score in the Sherrington et al. report, we chose to test this second categorization as well. We assumed a dominant mode of inheritance for the trait with a gene frequency of 0.0085 and a penetrance value of 0.86 and 0.76 for DOMSSF and DOMSS, respectively. A penetrance of 0.001 was fixed for the normal homozygote. Similarly to the Sherrington et al. report, age correction was not applied. The allele frequencies at the loci of the three markers, calculated from two panels of unrelated healthy individuals randomly chosen, were not found to differ significantly from those reported in previous reports. The heterogeneity of linkage data was assessed by two different tests: the first one, developed by Morton (1956) is called “the predivided sample test.” In this test, the total family sample is subdivided into subsamples according to specific criteria. We also used the “admixture test”of Smith (1963) as implemented in the HOMOG program of Ott (1985). This test assumes that the sample contains two kinds of families: those in which the disease is determined at a locus linked to the marker in a proportion a and the other l-a, where the disease segregates independently of the marker. For Mendelian diseases the lod score method is the most efficient method to detect linkage and to provide an estimate of the recombination fraction between the trait locus and the marker locus. However, the use of this method is seriously hampered in psychiatric diseases since the lod score computation assumes parameters (such as gene frequency and penetrance) that are unknown in these genetically complex diseases (Clerget-Darpoux et al., 1986). In such a case, nonparametric tests of linkage are especially relevant. Therefore, we analyzed our data by the affected pedigree member method (Weeks and Lange, 1988) which does not require knowledge of the mode of inheritance of the disease and, with the use of identity-by-state relations, can extract information from distantly affected relatives.
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Results Lod Score Method. Table schizophrenia fringe (DQMSSF proximal region of the long arm the results of Sherrington et al. from probe pl05-599ha and 11
2 summarizes two-point lod scores between classification) and three markers located in the of chromosome 5. Clearly, we have not replicated ‘(1988); moreover, we can reject linkage at 12 CM CM from probe pl05-153rb. When two-point lod
Table 2. Two-point lod scores between schizophrenia fringe (DOMSSF classification) and markers of chromosome 5 in 28 families Probes
0
0.00
0.10
0.20
0.30 -0.10
0.03
0.40
105599ha
-12.49
-2.94
-0.82
105-i
-10.64
-2.31
-0.78
-0.19
-0.02
-3.33
-0.57
-0.02
0.08
0.02
53ra
105798rb
Note. The lod score is the log to the base 10 of the probability that a given set of data about genetrc recombination would arise by virtue of two loci being linked at a specified recombination fraction divided by the probability that the data would arise by nonlinkage.
scores are calculated under the DOMSS classification, results are similar and linkage can be rejected at 11 CM from probe pl05-599ha and 8 CM from probe ~105-153 rb (Table 3). The point to be underlined is that despite using the same genetic parameters as Sherrington et al., we rejected linkage at the location where they found their peak lod score (8 CM from pl05-599ha in the direction of pl05-153ra). The aim of this study was to replicate a previous finding. In contrast to other authors, we did not extend the study to investigate genetic linkage in the entire 5ql l-q13 region. In particular, we made no attempt to calculate multipoint linkage to define a precise exclusion map. The reason for this choice is that the mode of inheritance of the disease remains unknown. In this context, the choice of an incorrect genetic model to perform linkage analysis may lead to false exclusion of linkage (Clerget-Darpoux et al., 1986; Rice et al., 1989; Risch et al., 1989). As pointed out by Amos et al. (1991) multipoint linkage is particularly sensitive to any type of misspecification of the genetic model. In our opinion, multipoint linkage is an inaccurate approach to exclusionary mapping in genetically complex diseases.
Table 3. Two-point lod scores between schizophrenia spectrum (DOMSS classification) and markers of chromosome 5 in 28 families 0.10
0.20
0.30
-11.65
-2.38
-0.58
-0.02
pi 05-l 53ra
-8.04
-1.68
-0.47
-0.02
0.06
pi 05-798rb
-2.76
-0.63
-0.24
-0.09
-0.03
Probes pl05-599ha
0
0.00
0.40 0.04
Note. The lod score is the log to the base 10 of the probability that a given set of data about genetic recombrnation would arise by virtue of two loci being linked at a specified recombrnation fractron divided by the probabikty that the data would arise by nonlinkage.
Heterogeneity. Two analyses were performed to test the heterogeneity of linkage data. First, the heterogeneity of lod score values was assessed within our own sample. Using the HOMOG program, we found no significant evidence for hetero-
175 geneity among our 28 families under any of the clinical classifications considered. We therefore performed a second analysis by looking for significant differences between our families and those of Sherrington et al. by means of a “predivided sample test.” The asymptotic x2 values for the analyses performed under the DOMSSF classification with the markers p105-599ha and p105-153ra are 5.89 and 6.72, respectively, and allow the rejection of the null hypothesis of homogeneity with a statistical significance of 0.02 and 0.01, respectively (data concerning the p105798rb locus were not available in the Sherrington et al. report). Affected Pedigree Members Method. We used the computer package provided by Weeks and Lange (1988) to detect departure from independent segregation between markers and disease loci. Tables 4 and 5 present combined statistics for the three markers under the two clinical classifications. Since the statistic is based on the marker phenotypes of affected relatives and since the sharing of a rare marker allele between distantly affected relatives has more weight than the sharing of a common marker allele, the statistic includes different weighting factors based on allele frequency. Whatever the weighting factor used, in no case did the results indicate a statistically significant departure from independent segregation of marker alleles. We wish to point out, however, that this negative finding does not constitute proof of a lack of linkage since the power of the method to detect linkage given the size of our sample is unknown.
Table 4. Analysis by the affected pedigree members method: Combined statistics for the 28 families (DOMSSF classification) Function
Statistic
D
value
A marker 105599ha f(P) = 1
-0.15
NS
f(P) = 16
-0.75
NS
-1.02
NS
-0.95 -1.07
NS
f(P) = l/d?
f (PI = l/P
-0.97
NS
f(P) = 1 /P B marker 105-l 53ra f(P) = 1
NS
C marker 105798rb f(P) = 1
-1.99
NS
f(P) = 16
-1.89
NS
f(P) = 1 /P
-1.71
NS
Discussion Our failure to replicate the results of Sherrington et al. (1988) could reflect the use of an invalid genetic model. It is important to stress that the parameters used in lod score calculation by Sherrington et al. (and by ourselves) are at best highly speculative. Several aspects of the original report of Sherrington et al. have been widely criticized. It has been argued, for example, that the model used does not fit well with the epidemiological data, that the definition of affected status is very
176
Table 5. Analysis by the affected pedigree members method: Combined statistics for the 28 families (DOMSS classification) Function
Statistic
D
value
A marker 105-599ha f(P) = 1
-0.82
NS
f(P) = l/Ji;
-1.42
NS
-1.21
NS
f(P) = 1
-0.88
NS
f(P) = l/Jis
-0.77
NS
-0.77
NS
f(P) = 1
-1.89
NS
f(P) = l/Ji;
-1.56
NS
f(P) = l/P
-1.36
NS
f(P) = l/P B marker 105-l 53ra
f(P) = 1 /P C marker 105-798rb
unusual, and that the absence of age correction in lod score computation is incorrect. In the presence of linkage, misspecifying genetic parameters in lod score analysis may lead to divergent results between studies (MacLean et al., 1975), especially when different ascertainment schemes have been used. When a valid genetic model is used, the conclusions of lod score analysis do not depend on the mode of ascertainment of families. However, if an invalid genetic model is used, heterogeneity of linkage data may be falsely suggested when ascertainment differs between two samples. There is a difference in ascertainment strategies between the two studies (at least two living first degree schizophrenic relatives in one case, multiple subjects affected in at least three generations in the other case), but it seems unlikely that such a minor variation could account for the significant heterogeneity we found. Moreover, the lack of evidence for linkage when performing a simultaneous nonparametric test does not support the hypothesis of a false-negative result in the present report. We need to consider a second possibility to explain these discordant resultsnamely, true genetic heterogeneity. It is conceivable that a susceptibility locus located on chromosome 5 might account only for a subset of families. However, when considering this fact, one might wonder which factors may cause this heterogeneity to be present not within each sample (i.e., the Sherrington et al. sample and our own sample) but between the two samples. The procedures followed in the collection of clinical information and the diagnostic criteria used were basically the same in the two studies. In particular, no bipolar illness was found in the two samples. If differences in methodological procedures are not responsible, what is the basis of these discordant findings? An ethnic explanation (a rare gene segregating in Iceland and United Kingdom, but not in other parts of the world) may be invoked. However, this possibility has already been discussed and rejected by McGuffm et al. (1990). In this context, as stressed by Owen et al. (1990), it is relevant to ask “why Sherrington et al. and not other groups were able to select families with a defect in chromosome 5.” This question remains unresolved. Finally, we have to consider the possibility that the findings of Sherrington et al.
177
may represent a false-positive result. We (Clerget-Darpoux et al., 1990) and others (Weeks et al., 1990; Risch, 1991) have shown that the significance of a lod score value of 3 is difficult to assess in linkage studies of a genetic marker and a complex disease, especially when multiple tests have been performed (several markers, several clinical classifications, and several genetic models). In such cases, the Type 1 error is no longer negligible and the probability of obtaining a lod score value exceeding 3 despite independent transmission of the disease and the marker increases substantially. Weeks et al. (1990) have reanalyzed the data of Sherrington et al. (1988), taking into account the inflation of lod scores due to maximization over models. They proposed to deflate the initial maximum lod score from approximately 1 unit, which still gives a significant result. It is important to stress that this estimate was strictly based on the reanalysis of the data published by Sherrington et al. Thus, the question Weeks et al. have investigated is the probability of falsely detecting linkage within one study. However, in attempts to deal with the problem of multiple tests, the correct approach is to assess the probability of obtaining a lod score above 3 by chance, taking into account all linkage tests actually performed. When many laboratories are involved in conducting similar studies with many markers, it is indeed very difficult to assess the exact number of tests performed. Thus, it seems to us that the adjustment proposed by Weeks et al. is an underestimate. In conclusion, it should be emphasized that true replication studies, performed with the same genetic models, the same diagnostic classifications, and the same genetic markers for which a positive linkage has been reported are necessary to be confident of the existence of a linkage in genetically complex diseases. Clearly, without such a replication, a positive finding is not sufficient to establish the presence of a susceptibility locus, no matter how large the initial reported lod score (Risch, 1990). Acknowledgments. The authors thank D. Samolyk and B. Henrickson for laboratory assistance. They also thank those colleagues who helped in the identification of families. This work was supported by INSERM grants (CRE 88-80-17 and reseau 48-80-l 1).
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178 Clerget-Darpoux, F., and Bonaiti-Pellie, C. Strategies based on marker information for the study of human diseases. Annals of Human Genetics, 56: 145-153, 1992. Clerget-Darpoux, F.; Bonaiti-Pellie, C.; and Hochez, J. Effects of misspecifying genetic parameters in lod score analysis. Biometrics, 42:393-399, 1986. Crowe, R.R.; Black, D.W.; Wesner, R.; Andreasen, N.; Cookman, A.; and Roby, J. Lack of linkage to chromosome 5ql l-q13 markers in six schizophrenia pedigrees. Archives of General Psychiatry, 48:357-361, 1991. Detera-Wadleigh, S.D.; Goldin, L.R.; Sherrington, R.; Encio, I.; De Miguel, C.; Berrettini, W.; Gurling, H.; and Gershon, E.S. Exclusion of linkage to 5qll-13 in families with schizophrenia and other psychiatric disorders. Nature, 340:391-393, 1989. Fyer, A.J.; Endicott, J.; Mannuzza, S.; and Klein D.F. Schedule for Affective Disorders and Schizophrenia-Lifetime Anxiety Version. New York: New York State Psychiatric Institute, 1985. Gottesman, I.I., and Shields, J. Schizophrenia: The Epigenetic Puzzle. Cambridge: Cambridge University Press, 1985. Gusella, J.F.; Wexler, N.S.; and Conneally, P.M. A polymorphic DNA marker genetically linked to Huntington’s disease. Nature, 306:234-238, 1983. Kaufmann, C.; DeLisi, L.E.; Lehner, T.; and Gillian, C. Physical mapping, linkage analysis of a putative schizophrenia locus on chromosome 5. Schizophrenia Bulletin, 15:441-452, 1989. Kennedy, J.L.; Giuffra, L. A.; Moises, H. W.; Cavalli-Sforza, L.L.; Pakstis, A.J.; Kidd, J.R.; Castiglione, C.M.; Sjogren, B.; Wetterberg, L.; and Kidd, K.K. Evidence against linkage of schizophrenia to markers on chromosome 5 in a northern Swedish pedigree. Nature, 336:167169, 1988. Lathrop, G.M.; Lalouel, J.M.; Jullier, C.; and Ott, J. Multilocus linkage analysis in humans: Detection of linkage and estimation of recombination. American Journal of Human Genetics, 37:482-498, 1985. Leboyer, M.; Jay, M.; d’Amato, T.; Campion, D.; Guilloud-Bataille, M.; Hillaire, D.; Drouet, A.; Lepine, J.P.; Bois, E.; and Feingold, J. Subtyping familial schizophrenia: Reliability, concordance, and stability. Psychiatry Research, 34:77-88, 1990. MacLean, C.J.; Morton, N.E.; and Rew, R. Analysis of family resemblance: IV. Operational characteristics of segregation analysis. American Journal of Human Genetics, 27:365-384, 1975. Maniatis, T.; Fritsch, E.F.; and Sambrook, J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. McGue, M.; Gottesman, 1.1.; and Rao, D.C. Resolving genetic models for the transmission of schizophrenia. Genetic Epidemiology, 2:99-l 10, 1985. McGuffin, P.; Sargeant, M.; Hetti, G.; Tidmarsch, S.; Whatley, S.; and Marchbanks, R.M. Exclusion of a schizophrenia susceptibility gene from the chromosome 5q I l-p13 region: New data and a reanalysis of previous reports. American Journal of Human Genetics, 471524-535, 1990. Merikangas, K.R.; Spence, M.A.; and Kupfer, D.J. Linkage studies of bipolar disorder: Methodologic and analytic issues. Archives of General Psychiatry, 46: 1137-l 141, 1989. Morton, N.E. The detection and estimation of linkage between the genes for elliptocytosis and the Rh blood type. American Journal of Human Genetics, 8:80-96, 1956. Neitzel, H. A routine method for the establishment of permanent growing lymphoblastoid cell lines. Human Genetics, 73:320-326, 1986.
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Failure to Replicate Linkage Between Chromosome 5qll -ql3 Markers and Schizophrenia in 28 Families Dominique Campion, Thierry d’Amato, Hassan Laklou, Olivier Sabate, Maurice Jay, Marion Leboyer, Alain Malafosse, Philip Gorwood, Marie Claude Babron, Dominique Hillaire, Franqoise Clerget-Darpoux, Gilles Waksman, and Jacques Mallet Received January 7, 1992; revised version received May 26, 1992; accepted September 5, 1992.
Abstract. Sherrington et al. (1988) reported linkage between markers located on the 5ql l-q13 region of chromosome 5 and schizophrenia in five Icelandic and two British families. To date, however, all attempts to replicate the initial finding have failed. Using three markers of chromosome 5, we have studied 28 additional French pedigrees. When our data were analyzed both with parametric (i.e., lod scores) and nonparametric methods, we found no evidence of linkage. Thus, we were unable to replicate the earlier report by Sherrington et al. Key Words. Genetic analysis, molecular genetics, affected-pedigree member method.
Despite significant evidence from adoption and twin studies that genetic factors play a role in the etiology of schizophrenia (Gottesman and Shields, 1985), the mode of inheritance of the disease remains elusive (McGue et al., 1985). Segregation analyses have yielded ambiguous results. Basically, the methodology of such studies suffers from intrinsic limitations-for example, the oversimplification of the models used and the assumption of genetic homogeneity (Baron, 1986). Advances in tiolecular genetics offer the opportunity to develop an alternative strategy for the detection of genetic risk factors. Indeed, the linkage approach, which studies the cosegregation between DNA markers and diseases within pedigrees, is a powerful tool to characterize susceptibility genes. This strategy has already achieved success in Mendelian diseases such as cystic fibrosis (Riordan et al., 1989) or Huntington’s chorea (Gusella et al., 1983). With the report by Sherrington et al. (1988) of linkage between schizophrenia and genetic markers located on the long arm of chromosome 5, it seemed that a gene might have been identified that predisposes to a genetically complex disease. Unfortunately, subsequent investigators have failed to detect linkage with chromosome
Dominique Campion, M.D., Marion Leboyer, M.D., Ph.D., Philip Gorwood, M.D., Marie Claude Babron, Ph.D., and FranCoise Clerget-Darpoux, Ph.D., are at the “Unit& de Recherche d%pidtmiologie G&nttique”(INSERM U 155), Chateaude Longchamp, 75016 Paris. Maurice Jay, M.D., is at Hbpital St Paul, La Reunion. Dominique Hillaire, Ph.D., is at “Genethon,” Evry. Thierry d’Amato, M.D., Hassan Laklou, Research Assistant, Olivier Sabate, M.D., Alain Malafosse, M.D., Gilles Waksman, Ph.D., and Jacques Mallet, Ph.D., are at “Laboratoire de Neurobiologie MoKzculaire et Cellulaire”(CNRS), 91190 Gif_sur Yvette. (Reprint requests to Dr. D. Campion, Centre Hospitalier Specialis& rue P. Eluard, Bolte Postale 45, 76301 Sotteville-l&s-Rouen, France.) 0165-1781/92/$05.00
@ 1992 Elsevier Scientific
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Ireland
Ltd
172 5ql l-q13 markers (Kennedy et al., 1988; Detera-Wadleigh et al., 1989; Kaufmann et al., 1989; St. Clair et al., 1989; Aschauer et al., 1990; McGuffin et al., 1990; Crowe et al., 1991). It could be argued that some of these studies (Kennedy et al., 1988; Detera-Wadleigh et al., 1989; St. Clair et al., 1989) are not true “replications” of the study of Sherrington et al. (1988) since the diagnostic procedures and the genetic parameters of the original report were not used. However, the models used in the various studies are similar enough to suggest that differences in parameter values cannot account for such discordant results. The contention that a susceptibility locus for schizophrenia can be excluded from chromosome 5qll-q13 and that the positive results of Sherrington et al. occurred by chance (McGuffin et al., 1990) is controversial (Amos et al., 1991). As the use of the lod score’ method in genetically complex diseases raises many methodological questions (Merikangas et al., 1989; Baron et al., 1990), it seemed appropriate to conduct a new replication study that combined the lod score analysis and a nonparametric approach (i.e., the affected pedigree member method; Weeks and Lange, 1988). To conduct this replication study, we therefore collected new multiplex pedigrees but found no evidence of linkage between schizophrenia and chromosome 5 markers. Methods Psychiatric admissions were systematically Family Ascertainment and Diagnoses. screened in two locations: the city of Rouen (France) and the island of la Rtunion (Indian Ocean). Details about ascertainment and diagnostic procedures are given elsewhere (Leboyer et al., 1990). In brief, families were ascertained on the basis of possessing at least two first degree relatives with a diagnosis of schizophrenia. Pedigrees showing evidence of assortative mating between schizophrenic subjects were discarded. Paternity was confirmed by human leukocyte antigen and red cell typing. Two semistructured interviews were used in the clinical assessment of probands and available relatives: the French translations of the Schedule for Affective Disorders and Schizophrenia-Lifetime Version (SADS-LA; Fyer et al., 1985) and the Schedule for Schizotypal Personalities (SSP; Baron et al., 1981). In addition, hospital charts were consulted. Best-estimate diagnoses according to DSM-III criteria (American Psychiatric Association, 1980) were made by two raters without knowledge of pedigree structure. Marker status was determined without knowledge of clinical diagnosis. Twenty-eight families (14 in metropolis and 14 in la Reunion) were included in this study. In these 28 families, there were 91 cases of schizophrenia, 3 cases of schizoaffective disorders, and 11 cases of schizophrenia spectrum disorders (schizotypal and paranoid personalities). Five individuals were classified as “unknown phenotype” and 21 as “other psychiatric disorders” (including 8 diagnosed as suffering from alcohol abuse, 4 from major depression, 3 from phobic disorder, 1 from obsessive-compulsive disorder, 3 from mental retardation (mild), 1 from dysthymic disorder, and 1 from somatization disorder). No individuals afflicted with bipolar disorder were found in our sample. Pedigrees are available from the authors on request. Restriction Fragment Length Polymorphism Typing. Venous blood samples were obtained from subjects and cell lines were estabished by Epstein-Barr virus transformation of fl lymphocytes (Neitzel et al., 1986). High molecular weight DNA was extracted from cultured I. The lod score = the log to the base IO of the probability that a given set of data about genetic recombination would arise by virtue of two loci being linked at a specified recombination fraction divided by the probability that the data would arise by nonlinkage.
173 cells, digested with Msp 1 and Taq 1 restriction enzymes, electrophoretically separated on agarose gel and transferred to nylon membranes by standard methods (Maniatis et al., 1982). The filters were hybridized with J2P radiolabeled probes. Locus D5 S76 was defined by a Taq 1 polymorphism detected by probe p105-599ha, and loci D5 S39 and D5 S78 were defined by Msp 1 polymorphisms detected by probes p105-153ra and p105-798rb, respectively. Table 1 provides details about the probes used, and the length and frequencies of the fragments recognized. On the basis of analysis of the families from the Centre d’fitude du Polymorphisms Humain (Weiffenbach et al., 1991), the order and approximate recombination distances between the markers are: p105-599ha (19 CM) p105-153rb (12 CM) p105-798ha.
Table 1. Chromosome 5 markers Marker 105~599ha
105-153ra 105-79&b
(locus)
Location
Polymorphism
5q12-14
Taq 1
5q12-14 5q12-14
Mspl Mspl
Alleles (kb)
Frequencies
al = 17.00
0.32.
a2 = 14.00
0.16
a3 = 10.00
0.50
a4 =
0.02
9.00
bl = 8.70
0.33
b2 = 5.80
0.67
cl = 2.30
0.57
c2=
0.43
1.80
Linkage Analysis. Two-point linkage analyses were performed with MLINK (Lathrop et al., 1985) using the same clinical classifications and genetic parameters as Sherrington et al. (1988) To define the pathological phenotype, we first used the clinical classification that gave the maximum lod score in the Sherrington et al. report. In this classification, called DOMSSF, all subjects with a psychiatric diagnosis were scored as affected. However, since this categorization is very unusual and since the more conventional DOMSS classification (which includes individuals with schizophrenia and schizophrenia spectrum disorders) also gave a positive lad score in the Sherrington et al. report, we chose to test this second categorization as well. We assumed a dominant mode of inheritance for the trait with a gene frequency of 0.0085 and a penetrance value of 0.86 and 0.76 for DOMSSF and DOMSS, respectively. A penetrance of 0.001 was fixed for the normal homozygote. Similarly to the Sherrington et al. report, age correction was not applied. The allele frequencies at the loci of the three markers, calculated from two panels of unrelated healthy individuals randomly chosen, were not found to differ significantly from those reported in previous reports. The heterogeneity of linkage data was assessed by two different tests: the first one, developed by Morton (1956) is called “the predivided sample test.” In this test, the total family sample is subdivided into subsamples according to specific criteria. We also used the “admixture test”of Smith (1963) as implemented in the HOMOG program of Ott (1985). This test assumes that the sample contains two kinds of families: those in which the disease is determined at a locus linked to the marker in a proportion a and the other l-a, where the disease segregates independently of the marker. For Mendelian diseases the lod score method is the most efficient method to detect linkage and to provide an estimate of the recombination fraction between the trait locus and the marker locus. However, the use of this method is seriously hampered in psychiatric diseases since the lod score computation assumes parameters (such as gene frequency and penetrance) that are unknown in these genetically complex diseases (Clerget-Darpoux et al., 1986). In such a case, nonparametric tests of linkage are especially relevant. Therefore, we analyzed our data by the affected pedigree member method (Weeks and Lange, 1988) which does not require knowledge of the mode of inheritance of the disease and, with the use of identity-by-state relations, can extract information from distantly affected relatives.
174
Results Lod Score Method. Table schizophrenia fringe (DQMSSF proximal region of the long arm the results of Sherrington et al. from probe pl05-599ha and 11
2 summarizes two-point lod scores between classification) and three markers located in the of chromosome 5. Clearly, we have not replicated ‘(1988); moreover, we can reject linkage at 12 CM CM from probe pl05-153rb. When two-point lod
Table 2. Two-point lod scores between schizophrenia fringe (DOMSSF classification) and markers of chromosome 5 in 28 families Probes
0
0.00
0.10
0.20
0.30 -0.10
0.03
0.40
105599ha
-12.49
-2.94
-0.82
105-i
-10.64
-2.31
-0.78
-0.19
-0.02
-3.33
-0.57
-0.02
0.08
0.02
53ra
105798rb
Note. The lod score is the log to the base 10 of the probability that a given set of data about genetrc recombination would arise by virtue of two loci being linked at a specified recombination fraction divided by the probability that the data would arise by nonlinkage.
scores are calculated under the DOMSS classification, results are similar and linkage can be rejected at 11 CM from probe pl05-599ha and 8 CM from probe ~105-153 rb (Table 3). The point to be underlined is that despite using the same genetic parameters as Sherrington et al., we rejected linkage at the location where they found their peak lod score (8 CM from pl05-599ha in the direction of pl05-153ra). The aim of this study was to replicate a previous finding. In contrast to other authors, we did not extend the study to investigate genetic linkage in the entire 5ql l-q13 region. In particular, we made no attempt to calculate multipoint linkage to define a precise exclusion map. The reason for this choice is that the mode of inheritance of the disease remains unknown. In this context, the choice of an incorrect genetic model to perform linkage analysis may lead to false exclusion of linkage (Clerget-Darpoux et al., 1986; Rice et al., 1989; Risch et al., 1989). As pointed out by Amos et al. (1991) multipoint linkage is particularly sensitive to any type of misspecification of the genetic model. In our opinion, multipoint linkage is an inaccurate approach to exclusionary mapping in genetically complex diseases.
Table 3. Two-point lod scores between schizophrenia spectrum (DOMSS classification) and markers of chromosome 5 in 28 families 0.10
0.20
0.30
-11.65
-2.38
-0.58
-0.02
pi 05-l 53ra
-8.04
-1.68
-0.47
-0.02
0.06
pi 05-798rb
-2.76
-0.63
-0.24
-0.09
-0.03
Probes pl05-599ha
0
0.00
0.40 0.04
Note. The lod score is the log to the base 10 of the probability that a given set of data about genetic recombrnation would arise by virtue of two loci being linked at a specified recombrnation fractron divided by the probabikty that the data would arise by nonlinkage.
Heterogeneity. Two analyses were performed to test the heterogeneity of linkage data. First, the heterogeneity of lod score values was assessed within our own sample. Using the HOMOG program, we found no significant evidence for hetero-
175 geneity among our 28 families under any of the clinical classifications considered. We therefore performed a second analysis by looking for significant differences between our families and those of Sherrington et al. by means of a “predivided sample test.” The asymptotic x2 values for the analyses performed under the DOMSSF classification with the markers p105-599ha and p105-153ra are 5.89 and 6.72, respectively, and allow the rejection of the null hypothesis of homogeneity with a statistical significance of 0.02 and 0.01, respectively (data concerning the p105798rb locus were not available in the Sherrington et al. report). Affected Pedigree Members Method. We used the computer package provided by Weeks and Lange (1988) to detect departure from independent segregation between markers and disease loci. Tables 4 and 5 present combined statistics for the three markers under the two clinical classifications. Since the statistic is based on the marker phenotypes of affected relatives and since the sharing of a rare marker allele between distantly affected relatives has more weight than the sharing of a common marker allele, the statistic includes different weighting factors based on allele frequency. Whatever the weighting factor used, in no case did the results indicate a statistically significant departure from independent segregation of marker alleles. We wish to point out, however, that this negative finding does not constitute proof of a lack of linkage since the power of the method to detect linkage given the size of our sample is unknown.
Table 4. Analysis by the affected pedigree members method: Combined statistics for the 28 families (DOMSSF classification) Function
Statistic
D
value
A marker 105599ha f(P) = 1
-0.15
NS
f(P) = 16
-0.75
NS
-1.02
NS
-0.95 -1.07
NS
f(P) = l/d?
f (PI = l/P
-0.97
NS
f(P) = 1 /P B marker 105-l 53ra f(P) = 1
NS
C marker 105798rb f(P) = 1
-1.99
NS
f(P) = 16
-1.89
NS
f(P) = 1 /P
-1.71
NS
Discussion Our failure to replicate the results of Sherrington et al. (1988) could reflect the use of an invalid genetic model. It is important to stress that the parameters used in lod score calculation by Sherrington et al. (and by ourselves) are at best highly speculative. Several aspects of the original report of Sherrington et al. have been widely criticized. It has been argued, for example, that the model used does not fit well with the epidemiological data, that the definition of affected status is very
176
Table 5. Analysis by the affected pedigree members method: Combined statistics for the 28 families (DOMSS classification) Function
Statistic
D
value
A marker 105-599ha f(P) = 1
-0.82
NS
f(P) = l/Ji;
-1.42
NS
-1.21
NS
f(P) = 1
-0.88
NS
f(P) = l/Jis
-0.77
NS
-0.77
NS
f(P) = 1
-1.89
NS
f(P) = l/Ji;
-1.56
NS
f(P) = l/P
-1.36
NS
f(P) = l/P B marker 105-l 53ra
f(P) = 1 /P C marker 105-798rb
unusual, and that the absence of age correction in lod score computation is incorrect. In the presence of linkage, misspecifying genetic parameters in lod score analysis may lead to divergent results between studies (MacLean et al., 1975), especially when different ascertainment schemes have been used. When a valid genetic model is used, the conclusions of lod score analysis do not depend on the mode of ascertainment of families. However, if an invalid genetic model is used, heterogeneity of linkage data may be falsely suggested when ascertainment differs between two samples. There is a difference in ascertainment strategies between the two studies (at least two living first degree schizophrenic relatives in one case, multiple subjects affected in at least three generations in the other case), but it seems unlikely that such a minor variation could account for the significant heterogeneity we found. Moreover, the lack of evidence for linkage when performing a simultaneous nonparametric test does not support the hypothesis of a false-negative result in the present report. We need to consider a second possibility to explain these discordant resultsnamely, true genetic heterogeneity. It is conceivable that a susceptibility locus located on chromosome 5 might account only for a subset of families. However, when considering this fact, one might wonder which factors may cause this heterogeneity to be present not within each sample (i.e., the Sherrington et al. sample and our own sample) but between the two samples. The procedures followed in the collection of clinical information and the diagnostic criteria used were basically the same in the two studies. In particular, no bipolar illness was found in the two samples. If differences in methodological procedures are not responsible, what is the basis of these discordant findings? An ethnic explanation (a rare gene segregating in Iceland and United Kingdom, but not in other parts of the world) may be invoked. However, this possibility has already been discussed and rejected by McGuffm et al. (1990). In this context, as stressed by Owen et al. (1990), it is relevant to ask “why Sherrington et al. and not other groups were able to select families with a defect in chromosome 5.” This question remains unresolved. Finally, we have to consider the possibility that the findings of Sherrington et al.
177
may represent a false-positive result. We (Clerget-Darpoux et al., 1990) and others (Weeks et al., 1990; Risch, 1991) have shown that the significance of a lod score value of 3 is difficult to assess in linkage studies of a genetic marker and a complex disease, especially when multiple tests have been performed (several markers, several clinical classifications, and several genetic models). In such cases, the Type 1 error is no longer negligible and the probability of obtaining a lod score value exceeding 3 despite independent transmission of the disease and the marker increases substantially. Weeks et al. (1990) have reanalyzed the data of Sherrington et al. (1988), taking into account the inflation of lod scores due to maximization over models. They proposed to deflate the initial maximum lod score from approximately 1 unit, which still gives a significant result. It is important to stress that this estimate was strictly based on the reanalysis of the data published by Sherrington et al. Thus, the question Weeks et al. have investigated is the probability of falsely detecting linkage within one study. However, in attempts to deal with the problem of multiple tests, the correct approach is to assess the probability of obtaining a lod score above 3 by chance, taking into account all linkage tests actually performed. When many laboratories are involved in conducting similar studies with many markers, it is indeed very difficult to assess the exact number of tests performed. Thus, it seems to us that the adjustment proposed by Weeks et al. is an underestimate. In conclusion, it should be emphasized that true replication studies, performed with the same genetic models, the same diagnostic classifications, and the same genetic markers for which a positive linkage has been reported are necessary to be confident of the existence of a linkage in genetically complex diseases. Clearly, without such a replication, a positive finding is not sufficient to establish the presence of a susceptibility locus, no matter how large the initial reported lod score (Risch, 1990). Acknowledgments. The authors thank D. Samolyk and B. Henrickson for laboratory assistance. They also thank those colleagues who helped in the identification of families. This work was supported by INSERM grants (CRE 88-80-17 and reseau 48-80-l 1).
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