- Email: [email protected]
Evidence against linkage of schizophrenia to chromosome 5q11-q13 markers in systematically ascertained families
BIOL PSYCHIATRY 1992;3! :83-94
83
Evidence against Linkage of Schizophrenia to Chromosome 5ql 1-q 13 Markers in Systematically Ascertained Families Joachim Hallmayer,* Wolfgang Maier, Manfred Ackenheil, Martha A. Ertl, Sabine Schmidt, Jtirgen Minges, Dirk Lichtermann, and Dieter Wildenauer
Ten pedigrees systematically ascertained in Germany were tested for linkage to chromosome 5ql l-q13. In order to replicate the previous report by Sherrington et al (1988), families with a bipolar family member were omitted from the lod score calculations, all diagnoses were based upon Research Diagnostic Criteria, and four different models of the affection status were calculated, including the model for which Sherrington et al calculated the highest lod scores. None of the families investigated showed a positive lod score. Using multipoint linkage analyses, we were able to exclude the region for which a positive linkage has been reported.
Introduction Family, twin, and adoption studies have indicated that the etiology of schizophrenia is genetic, at least in part. In 1988, evidence that a gene causing schizophrenia is located on chromosome 5ql 1.2-q13.3 was reported based on a schizophrenic uncle-nephew pair with partial trisomy of 5q (Bassett et al 1988). Sherrington et al conducted linkage studies with DNA markers spanning the proximal portion of the trisomy including the 5q11.2 breakpoint in families from Iceland and Britain (Gilliam et al 1989). They reported positive lod scores between schizophrenia and the probes p105-153Ra (D5S39) and plO5-599Ha (D5S76). Subsequent linkage studies (Aschauer et al 1990; Crowe et al 1991; Detera-Wadleigh et ai 1989; Kennedy et al 1988; McGuffin et ai 1990; St. Clair ct al 1989) excluded linkage to schizophlenia using the same probes as Sherrington et al, but in different families. In the study of Sherdngton et al (1988), lod scores reached much higher values when models using ~ broader definition of the affection status were tested. The highest loci score~ were calculated when family members who suffered from a variety of psychiatric
From the Departmentof Psychiatry, University of Mainz, Germany (JH, WM, JM, DL); and Department of Neurochemistry, Psychiatric Hospt'tel. University of Munich.,Gemtaay (MA, MAE, SS, DW). *Current address: Departmelltof Genetics, Stanford University School of Medicine, Stanford, CA. Address reprint requests to Dr. Joachim H~|lmayer,Departmentof Genetics, StanfordUniversitySchool of Medicine, Stanford, CA 94305. Received September 18, 1990; revised May 28, 1991. © 1992 Society o! Biologi,~alPsycbi,atry
t~06-3223/92/$05.00
84
BIOL PSYCHIATRY 1992;31:83-94
J. Hallmayeret al
disorders were also considered as affected in the analysis. The affection status also included diseases for which no higher prevalence in the relatives of schizophrenic probands has been described so far. This result suggested that a genetic predisposition for a variety of psychiatric diseases is transmitted by a single dominant gene. A major difference between the results of Sherdngton et al and some studies rejecting linkage (e.g., Detera-Wadleigh et al 1989; St. Clair et al 1989) is that in the latter studies, families with a bipolar family member are included in the analysis but the) had been omitted in the initial study. It was therefore suggested by Gurling et al (1989) that inclusion of bipolar disorder in the definition of the affection status resulted in nonreplication of th~ positive linkage. Different diagnostic criteria may also have led to differences in the studies. Diagnoses of the Swedish kindred (Kennedy et al 1988) were based upon DSMIll criteria whereas Sherdngton et al applied Research Diagnostic Criteria (RDC). In previous studies, families characterized by a high number of affected relatives were selected for linkage studies; it is not evident that they were selected in a systematic manner. This fact~ together with the remoteness of the area where the most informative families were derived, make replication of their study very difficult (Weeks et al 1990). In the present study, all families with a minimum number of affected fatally members according to family history were included in the linkage study. All families of patients treated for schizophrenia at the Psychiatric University Hospital in Mainz and Munich were asked to participate. If, according to family history, at least one first-debree relative and another first-degree or second-degree relative showed symptoms of schizophrenia or schizoaffective disorder a personal psychiatric imerview was carded out and blood was drawn from all available family members. Our recruitment of families was based only on the number of affected family members diagnosed as schizophrenic or schizoaffective by family history. All families fulfilling the criteria mentioned above were recruited for the linkage analysis regardless of the final diagnoses. Linkage studies were carried out on these families with markers in the region studied by Sherrington et al (1988). In order to make our study comparable to the study by Sherrington et al, all families with a bipolar family member were omitted from the loci score calculations, diagnoses were made according to RDC, and four different models of the affection status were calculated, including the model for which Sherrington et al calculated the highest lod scores.
Material and Methods Ten families of patients suffering from chronic schizophrenia, with at least two relatives showing symptoms of schizophrenia or schizoaffective psychosis according to family history, were included in the study. In none of these families was a person diagnosed as having bipolar disorder. Pedigree charts of these 10 families arc shown in Figurc 1. Best estimate diagnoses (Leckman et al 1982) of probands and all participating relatives were made by review of two interviews conducted by specially trained cesearch assistants, by all available medical records, and by information given by rclatives. The types of interviews used were the Schedule for Affcctive Disorders and Schizophrenia-Lifetime Version (SADS-LA) (Fyer et al 1~85) and the Structured Clinical Interview for DSM111 diagnoses-Personality Disorders,; (Spitzer and Williams 1985). The best estimates of two raters were comparcd. If there was no agreement, the individual files were reviewed on a case-by-case basis and a consensus diagnosis was established. All diagnoses were based upon RDC (Spitzer et al 1978). Lymphocytes from patients and participating rclatives were immortalized and cultured.
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY 1992;31:83-94
85
Family I
S
S
S
S
Family i?.0
S
a
a
S
S
•
S
Family 3
~
S
S
S
S
S
Family 4
8
S
m
S
Family $
S
S
~
Family 6
i
m
a
m
m
i
i
S
S
S
S
•
S
m
Family 7
S
S
8
S
Family 8
i
Family 9
S
S
S
i
ean~, lo
$
$
m
$
$
El 0 unaffected I • sch~ i • scldzom%cfivedisorder
g Q mjor delmmive disorder Figure 1. Pedigrees with several affected family members by schizophrenia. Asterisk indicates family members from whom blood has been drawn and genoUipings have been carried out with the markers described in the te~t,
86
J. Hallmayer et al
BIOL PSYCHIATRY 1992;3! :83-94
Table I. Probes, Loci, and Polymorphisms on Chromosome 5 Typed in the German Pedigrees Allele Locus
Pro~
Enzyme
Sym
D5S76
p105-5~)Ha
TaqI
D5S6
M4
BamHI
D5S39
p105-153~a
MspI
D5S78
p 105-7981~b
Mspl
AI A2 A3 A! A2 A3 AI A2 AI A2
Size (kb)
Freq
17.0 14.0 10.0 11.0 9.6 7.6 8.7 5.8 2.3 1.8
0.32 O. 16 0.52 0.33 0.52 O. 15 0.33 0.67 O.57 0.43
Williamsonct al 1990.
DNA was extracted tram cell lines, and 5-10 Ftg were digested overnight with the appropriate restriction enzymes to reveal the restriction enzyme polymorphism (Table 1). The digested DNA was electrophoresed on 0.8% agarose gel for 10--16 hr. The DNA was denatured and transferred to a nylon filter according ~o the method described by Southern (1975). Probes were labeled with 32p-dATP by the random primer oligonucleotide method (Feinb~rg and Vogelstein 1983). After prehybridization, the filters were hybridized at 65°C under standard conditions (Sambrook et al 1989). Washed filters were exposed to a Kodak XAR-film at -70°C. Films were developed after 3 and 14 days. Genotype assignment was done by researchers who were blind to the affection status of the subjects. Four probes were used in the present study (Table I):p105-153Ra identifying the locus D5S39, M4 (D5S6), p105-599Ha (D5S76), and p105-798Rb (D5S78) (Kiddet al 1989; Williamson et al 1990). Sherrington et al (1988) reported positive Iod scores using the probes p105-153Ra (D5S39) and p105-599Ha (D5S76). M4 (D5S6) is located between these two probes (Figure 2) and p105-798Rb (D5S78) is 9 centimorgans distal from p105153Ra (D5S39). Schizophrenia was assumed in the analysis to have a dominant mode of inheritance with incomplete penetrance, as in the study by Sherrington et al. Estimation of the penetrance parameters was based upon a large family study carried out in the area of Maiaz (Maier et al, unpublished data 1991). Lod scores were calculated under four different models of the phenotype (Table 2). IVlodel I restricted the affection phenotype to schizophrenia and schizoaffective psychosis. The phenotype of nonaffected individuals
0.07 |_
, D5876
'
0.06 t D5S6
'
0.09 I D5S39
'
I D5S78
Figure 2. Map of chromosome 5 showing the rv:gion of the typed markers (Giuffra et al 1989). Distances a-e given in Morgans.
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY 1992;31:83-94
87
Table 2. Models Defined for Calculations of Lod Scores Model
Affection status
Model !
Schizophrenia Schizoaffective disorder Schizophrenia Schizoaffective disorder Schizophrenia Schizoaffective disorder Schizotypal personality Disorder Major depressive disorder Sch-~phrenia Schizoaffective disorder Schizotypal personality Bim31ar disorder Major depressive disordel, Minor depressive disorder Alcoholism Anxiety disorders
Model 2 Model 3
Model 4
Nonaffection status
Penetrance (%)
Unknown Known
55
Known
77
Known
85
was tre:~:ed as unknown. In this model, the unaffected do not ¢on~bute to the lod score. This means that neither a false-negative diagnosis nor incorrect estimation of the penetrance affect the calculation of the loci score (Goldin and Martinez 1989). In model 2, a penetrance of 55% was assumed for schizophrenia and schizoaffective disorder. Model 3 (assuming 77% penetrance) included additionally all cases of major affective disorders and schizotypal personality disorde~,s as affected. Anxiety disorders, minor affective disorders, and alcoholism as well as all cases of model 3 were included in model 4 (assuming 85% penetrance). Model 4 is the model Sherrington et al (1988) used in obtaining the highest positive lod scores. Based upon the age of onset of schizophrenia and affective disorders, seven agedependent liability classes were established, for which the penetrance is raised until the maximum value is reached (55% for model 2, 77% for model 3, and 86% for model 4). For iod score calculations, the programs MLINK and LINKMAP of the program package LINKAGE (Version 4.91) (Lathrop and Lalouel 1984; Lathrop et al 1985) were used. Multipoint analysis was carried out against a fixed map of the loci D5S76, D5S6, D5S39, and D5S78 (Giuffra et al 1989) (Figure 2). Two four-point linkage maps were calculated: (1) D5S76-D5S6-D5S39 and (2) D5S76-D5S6-D5S78. Results Altogether, diagnoses and genotypes were available from 138 family members (Figure l) of l0 families. Of TIhese, 16 were diagnosed as having schizophrenia and another I l as schizoaffective. Jtn ~ddition, 25 family members had a major depressive disorder. Panic disolder was found in one family member. Personality disorders were diagnosed in three of the participating individuals; none of them belonged to the schizotypa! type. In one family (family 10), major depressive disorder was found to be bilateral. In the study by Sherri~igton et al 1988, only families with a single source of the illness were collected.
88
BIOL PSYCHIATRY 1992;31:83-94
J. Hallmayc.ret al
As the data of markers in the disease pedigrees should be consistent with published data from the reference families (Gershon et al 1990), we calculated two muitipoint analyses between the markers with the distances given by (3iuffra et al (1989). D5S76D5S6-D5S39 resulted in a loglikelihood difference of 7.7 and D5S76-D5S6-D5S78 of 6.8 with the recombination fractions given by Giuffra et al. Two-point lod scores between schizophrenia and the tested markers are shown in Tables 3 and 4. All lod scores are negative for recombination fractions of 0% and 5% except for p105-798Rb (D5S78) in model 1. Tight linkage between M4' (D5S6) and schizophrenia can be excluded in models 3 and 4. One problem with calculating lod scores for schizophrenia is that the mode of transmission, and therefore the true penetrance, is urLknown. The unaffected status can also b~ difficult to evaluate. Therefore, the preferred method is to use affected cases only, excluding the possibility of false-negative cases as in model 1 (Ott 1990). For this model, two-point iod scores are - i. i0 at 0% recombination for D5S76 and - i.29 for D5S6 at 0% recombination. Using multipoint analyses, all loglikelihood differences are less than - 2 in the region between D5S76 and D5S39 in models 2, 3, and 4 (Figures 3 and 4). Tkerefore, the entire region between D5S76 and D5S39 can be excluded for linkage. In models 3 and 4 the whole region between D5S39 and D5S7~ can be also excluded. Altogether, the exclusion area in model 2 is about 20 centimorgans, in model 3 ~bout 30, and in model 4 about 40.
Discussion Our results are in concordance with the reports from several laboratories (Aschauer et al 1990; Crowe et al 1991; Detera-Wadleigh et al 1989; Kennedy et al 1988; McGuffin et ai 1990; St. Clair et al 1989). So far all studies have failed to replicate the pos!tive findings of Sherrington et al (1988). Besides pure chance, failure to replicate the positive results has been explained by genetic heterogeneity, differences in the population studied, differences in the definition of the affection status, and the ascertainment of families (Baron et al 1990; Gershon et al 1990; Owen et al 1990). Genetic heterogeneity means different mutant alleles give rise to the same phenotype and are segregating independently in populations° As pointed out by Risch (1990), however, to conclude genetie heterogeneity in the absence of a Mendelian inheritance pattern, linkage evidence to a given chromosomal region must be replicated by at least one additional laboratory and replicated evidence for linkage to a second chromosomal region must be demonstrated. As neither criterion has been met, the contradictory results could be due either to nonreplication or to genetic heterogeneity. If genetic heterogeneity is a~sumed, negative iod scores have to be interpreted with caution as a lod score of less than - 2 as criterion is only valid in the case of homogeneity. None of the families studied by our ~ 0~p ~s~',ted in positive lod scores that would have indic~ted heterogeneity. McGuffin et al (1990) carried out linkage analyses with the data by Sherrington et al (1988) together with the n~gative results published so far. They found no evidence for heterogeneity among families in the stcdies showing no evidence for linkage and concluded that true heterogeneity is unlikely to exist. All evidence for heterogeneity is derived from the one positive finding by Sherrington et al. Given the large number of families showing no linkage, it is obvious that the number of linked families is quite small (Generate! and Kidd !99!).
BIOL PSYCHIATRY 1992;31:83-94
L i n k a g e to C h r o m o s o m e M a r k e r s in S c h i z o p h r e n i a
89
T a b l e 3. T w o - p o i n t L o d s c o r e s b e t w e e n S c h i z o p h r e n i a a n d M a r k e r s f o r C h r o m s o m e 5q I 1-13 D5S76 Recombination fraction Family
0.0
0.05
0.1
0.20
0.30
0.40
7
- !.77 -0.64 0.00 0.0! -0.18 -0.01 0,00
-0.9! -0.47 0.00 0.01 -0.13 -0.01 0,00
-0.53 -0.35 0.00 0.00 -0.09 -0,01 0,00
-0.16 -0.17 0.00 0.00 -0.04 -0.01 0,00
-0.02 -0.07 0.00 0.00 -0.01 0.00 0,00
0.02 -0.02 0.00 0.00 0.00 0.00 0,00
8
0.00
0.00
0.00
0.00
0.00
0.00
9 10
0.00 - 0.20 - 2.78
0.00 - 0.16 - 1.67
0.00 - 0.12 - 1.09
0.00 - 0.07 - 0.45
0.00 - 0.03 - 0.14
0.00 - 0.01 - 0.01
-0.65 0.20 0.00 - 0.04 0.05 - 1.49 0.00 0.00 0.01 -0.04 - 1.96
-0.35 0.17 0.00 - 0.04 0.04 - 0.74 0.00 0.00 0.01 -0.03 - 0.93
-0.21 0.14 0.00 - 0.04 0.03 - 0.48 0.00 0,00 0.01 -0.02 - 0.56
-0.07 0.09 0.00 - 0.03 0.02 - 0.23 0.00 0.00 0.01 0.00 -0.22
- 0.02
0.04 0.00 - 0.02 0.01 - 0.09 0.00 0,00 0,01 0.00 - 0.08
- 0.01 0.01 0.00 - 0.01 0.00 - 0.02 0.00 (L00 t3,00 o.00 - qL02
!
- 0.45
2 3 4 5 6 7
-0.17 0.02 0.02 -0,21 - 0.02 -0.01
- 0.25 -0.13 0.02 0.01 -0,16 - 0.02 -0.01
- 0.14 -0.10 0.01 0.01 -0,12 - 0.02 -0,01
- 0.03 -0.05 0.01 0.00 -0,06 - 0.01 0.00
0.01 -0.02 0.00 0.00 - 0.03 -0.01 0.00 0.00 0.00 0,01 - 0,03
0.02 -0.01 0f~) 0.00 - 0.01 0.00 0 00 0.00 0.00 0,00 0,01
- 0.06
- 0.03 0.00 0.00 0.00 -0.01 0.00 0,00 0.00 0,00 - 0,01 -0,04
6
Sum D5S6 l 2 3 4 5 6 7 8 9 10 Sum D5839
8
0.00
0.00
0.00
0.00
9 10
- 0.02 0.03 -0.81
- 0.01 0.03 -0.53
- 0.01 0.02 -0.35
- 0.01 0.02 -0.13
I
- 0.30
2 3 4 5 6 7 8 9 10
0.00 0.02 - 0.06 -0,19 0.00 - 0,02 0.00 0,01 -0,21 - 0.75
- 0.23 0.00 0.02 - 0.05 -0,15 0,01 - 0,02 0.00 0,01 -0,~.7 - 0.57
- 0.17 0.00 0.01 - 0.04 -0,11 0,02 - 0,02 0.00 0,01 -0,13 - 0.43
- 0.10 0.00 0.01 - 0.02 -0,06 0,02 - 0,01 0,00 0,00 -0,07 - 0.23
Sum
D5S78
Sum
0.00 0.00 -0.01 -0.03 0.01 0,00 0.00 0.00 - 0,03 -0,11
Schizophrenia was treated as a dominant disease and the penetrance was assumed to be 55% (model 2).
90
J. H a l l m a y e r et al
BIOL PSYCHIATRY 1992;31:83-94
Table 4. Two-point Lod Scores between Schizophrenia and Chromosome 5 q l 1-13 Markers Recombination fraction Marker
Model
0.00
0.05
O. 10
0.20
0.30
0.40
D5S76
1 2 3 4 l 2 3 4
- 1, IO -2,78 -0.86 - 1.46 - 1,29 - 1,96 -3.47 - 3.29 - 0.09 -0.81 - 1.88 - 1.08 0,26 -0.75 - !.81 -2.62
- 0.62 - 1.67 -0.19 -0.70 -0.80 - 0.93 -2.23 -2.12 - 0.01 -0.53 - 1.25 -0.73 0.22 -0.57 - 1.39 -2.01
- 0.37 - 1.09 0,07 -0.31 -0.54 - 0,56 - 1.56 - 1.47 0.03 -0.35 -0.85 -0.48 O. 18 -0,43 - 1.05 - 1.50
- O. 12 -0.45 0.21 0,01 -0.25 - 0,22 -0,75 -0,68 0.05 -0.13 -0.37 -0.18 0.09 -0.23 -0.54 -0.75
- 0.02 -0.14 0.15 0.07 -0.10 - 0.08 -0.30 -0,26 0,04 -0,03 -0,13 -0.05 0.03 -0.11 -0.22 -0.30
O.O! -0.01 0.06 0,03 -0.02 - 0.02 -0.07 -0.05 0.02 0,01 -0.02 0.O0 0.00 -0.04 -0.05 -0.07
D5S6
D5S39
1
D5S78
2 3 4 I 2 3 4
Four different definitions of the affection status were calculated.
0 -1
l
-2 -3
mm
]J - 4 -5 m
-6 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Distance in Morgans Figure 3, Multipoint analyses o f schizophrenia against a fixed map o f DSS76-DSS6-D5S39. D 5 S 7 6 was set arbitrarily at 0,2 M on the map, The other markers were set according to genetic distances (Figure 2 and Giuffra et al 1989), Four different models of the affection status were calculated as defined in Table 2. Model ! (=) model 2 (n) model 3 (&); model 4 (A).
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY ! 992;31:83-94
91
0 -1
§
-2
|
-3
c 0
-4 -5
¢/) tO
C1
-6 0
0.1
0.2
¢n ~
t43
~t[
a
0.3
0.4
0.5
0.6
0.7
0.8
Distance in Morgans Figure 4. Multipointanalyses of schizophrenia against a fixed map of DSS76-D5S6-DSS78. D5S76 was set arbitrarily at 0.2 M on the map. The other markers were set according to genetic distances (Figure 2 and Giuffra et al 1989). Four different models of the affection status were calculated as defined in Table 2. Symbols same as in Figure 3.
One reason for the presence of a rare gene in the families collected by Sherrington et al may be the remote area from which the families were collected; five families are of Icelandic origin. Iceland is genetically isolated from the rest of the world (Cavalli-Sforza et al 1991) and the population genetically closest to Icelanders is that of Norwegians. Therefore, it may be that the families collected by our group differ genetically from these Icelandic pedigrees. On the other hand, two families from the United Kingdom (one from Wales) studied by Sherrington et al (1988) also gave positive lod scores whereas in six other families of South Wales origin no li~'~kagecould be detected between chromosome 5 markers and schizophrenia (McGuffin et al 1990). Given these results, a specific Icelandic "schizophrenia gene" seems unlikely. The greatest difficulty in carrying out linkage analysis in psychiatric disorders is defining the affected phenotype and estimating the parameters describing the genetic model. Recently it was shown that maximization of the lod score (Clerget-Darpoux et al 1990) calculating different models of the affection status leads to an increased probability of false-positive results. The highest lod scores in the study of Sherrington et al were found by including a great variety of psychiatric diseases. The penetrance was estimated by maximizing the lod scores with increasing penetrance values. Th;~s model is the same as model 4 in our study with the exception that the gene frequency in our model is much higher. The penetrance values as well as the gene frequencies were fixed before calculating the Iod scores. Lowering the gene frequency to the values used by Sherrington et al results in even lower lod scores (data not shown).
92
J. Hallmayeret al
BIOL PSYCHIATRY 1992;31:83-94
0 -1 =o
-2 -3
-7
-8
0
0.1
0.2
0.3
0.4
0.5
0.6
Distance in M0rgans Figure 5. Multipoint anlayses of schizophrenia against a fixed map of DSS76-D5S6-DSS39. The calculations include five families in which at least 1 family member was diagnosed as bipolar. D5S76 was set arbitrarily at 0.2 M on the map, The other markers were set according to genetic distances (Figure 2 and Giuffra et al 1989), Four different models of the affection status were calculated as defined in Table 2. Symbols same as in Figure 3,
The determination that a person is truly affected presents a major problem in psychiatry. Most psychiatric diseases have a variable age of onset, and milder form of psychopathology may be considered normal. The preferred method of detecting linkage is to carry out an analysis only on affected subjects, while considering that the unaffected have unknown phenotypes. This "conservative" procedure does not decrease the ability to detect or exclude linkage; however, the sample size needed to detect and exclude linkage is increased (Goldin and Martinez 1989). Two-point lod scores for this model (model 1) were negative for D5S76, D5S39, and D5S6 but they did not reach the significance level of less than - 2. Five more families have been collected in which the diagnostic evaluation showed that at least one family member was diagnosed as being bipolm-. Inclusion of these five families in the calculations resulted in significantly negative lod scores (Figure 5) also for model ! as well as for models 2, 3, and 4. Whether families with bipolar disorder should be included in linkage studies of schizophrenia is controversial. The studies of Detera-Wadleigh et al (1989) and St. Clair et al (1989) both collected families with bipolar disorder, in a recent controlled family study it was shown that manic and schizophrenic syndromes were transmitted independently in families. Major depression, however, turned out to be an indic~or for a higher susceptibility tbr schizophrenia and bipolar disorder (Maier et al unpublished data 1991). Given these results, it can be speculated that two different genes--one a ,,usceptibility gene causing schizophrenic syndromes, one causing mania--both result in a higher vul-
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY 1992;31:83-94
93
nerability to unipolar depression. Inclusion of families with bipolar disorder would therefore result in two different genes causing unipolar depression. The detection of linkage for the broader definition of the phenotype would be difficult. On the other hand, restriction of the phenotype to a narrow definition of the phenotype should not decrease the chance of detecting linkage. Though we have applied a methodology similar to that used by Sherrington et al (1988), we were unable to find any evidence for dominant susceptibility gene for schizophrenia on chromosome 5q I l-q13 in our families from Germany. We wish to thank Prof. Dr. Mundt, Dr. Schleuning, and Dr. Minelli for their help in collecting and diagnosing the pedigrees. We also thank F. Schmidt, S. Scl~,wab, and U. Schoppe for preparation of probes and enzyme digestions. We are grateful to J. Mountain for he~ critical comments. All probes were received through ATCC. Dr. Hallmayer is supported by a fellowship of the Deutsche Forschungsgemeinschaft. This study was supported by the Deutsche Forschungsgemeinschafi grant numbers Wi 453/7-1 and Ma ! 142tl-3.
References Aschauer HN, Aschauer-Treiber G, Isenberg KE, et al (1990): No evidence for linkage between chromosome 5 markers and schizophrenia. Hum Hered 40:109-115. Baron M, Endicott J, Ott J (1990): Genetic linkage in mental illness. Limitations and prospects. Br J Psychiatry 157: 645--655. Bassett A J, Mc Gillivray BC, Jones BD, Pantzar JT (1988): Partial trisomy 5 cosegregating with schizophrenia. Lancet 9: 799-801. Cavalli-Sforza LL, Piazza A, Menozzi P (1991). Histmy and Geography of Human Genes. Princeton University Press. Clerget-Darpoux F, Babron M-C, and Bonaiti-Pellie C (1990): Assessing the effect of multiple linkage tests in complex diseases. Genet Epidemiol 7: 245-253. Crowe RR, Black DW, Wesner R, Andreasen NA, Cookman A, Roby J (1991): Lack of linkage to chromosome 5ql I-q13 markers in six schizophrenia pedigrees. Arch Gen Psychiatry. 48: 357-361. Detera-Wadleigh SD, Goldin LR, Sherrington R, et al (1989). Exclusion of linkage to 5ql 1-13 in families with schizophrenia and other psychiatric 0isorders. Nature 340: 391-393. Feinberg AP, Vogelstein B (1983): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1326-1333. Fyer AZ, Endicott Z, Mannuzza S, Klein DF (1985): Schedule for Affective Disorders and Schizophrenia, Lifetime Version (SADS.LA). New York: New York Psychiatric Institute. Gelernter J, Kidd KK (1991): The current status of linkage studies in schizophrenia. In McHugh PR, McKusick VA (eds.), Genes, Brain, and Behavior (New York: Raven Press, pp 137-152. Gershon ES, Martinez M, Goldin LR, Gejman PV (1990): Genetic mapping of common diseases: The challenges to manic-depressive illness and schizophrenia. Trends Genet 6: 282-287. Gilliam TC, Freimer NB, Kaufmann CA, et al (1989): Deletion map-"ag of DNA markers to region of chromosome 5 that cosegregates with schizophrenia. Genom -s 5: 940-944.
a
Giuffra LA, Kennedy JL, Pakstis AJ, et al (1989): A linkage group of ,., litarkers spanning >100 cM on chromosome 5 based on non-CEPH families. Cytogenet Cell Genet 51: 1004. Goldin LL, Martinez MM (1989): The detection of linkage and heterogeneity in nuclear families when unaffected individuals are considered unknown. Muhipoint Mapping and Linkage Based
94
BIOL PSYCHIATRY 1992;31:83-94
J. Hallmayer et al
Upon Affected Pedigree Members: Genetic Analysis Workshop 6. New York: Alan R. Liss, pp 195-200. Gurling HMD, Sherrington RP, Brynjolfsson J, et al (1989): Recent and future molecular genetic research into schizophrenia. Schizophr Bull 15: 373-382. Kennedy JL, Giuffra LA, Moises HW et al (1988): Evidence against linkage of schizophrenia to markers on chromosome 5 in a northern Swedish pedigree. Nature 336: 167-170. Kidd KK, Bowcock AM, Schmidke J, et al (1989): Report of the DNA committee and catalogs of cloned and mapped genes and DNA polymorphisms. Cytogenet Cell Genet 51: 622-947. Lathrop GM, Lalouel JM (1984): Easy calculations of lod scores and genetic risk on small comput.ers. Am J Hum Genet 36:460-465. Latlu'op GM, Lalouel JM, Julier C, Ott J (1985): Multilocus linkage analysis in humans: Detection of linkage and estimation of recombina'~ion. Am J Hum Genet 37: 482-498. Leckman, JF, Sholomskas D, Thompson D, Belanger A, Weissmann MM (1982): Best estimate of lifetime psychiatric diagnosis. Arch Gen Psychiatry 39: 879-883. McGuffin P, Sargeant M, Hetti G, Tidmarsh S, Whatley S, Marchbranks RM (1990): Exclusion of a schizophrenia susceptibility gene from the chromosome 5q I l-q 13 region: New data and a reanalysis of previous reports. Am J Hum Genet 47: 524-535. Ott J (1990): Genetic linkage and complex diseases: A comment. Genet Epidemiol 7: 35-36. Owen M, Crauford D, St Clair D (1990): Localization of a susceptibility locus for schizophrenia on chromosome 5. Br J Psychiatry 157: 123-127. Risch N (1990): Genetic linkage and complex diseases, with special reference t(~ psychiatric disorders. Genet Epidemiol 7: 3-16. Sambrook J, Fritsch EF, Maniatis T (1989): Molecular Cloning. A Laboratory Manual (ed 2). Cold Spring Harbor, NY: Laboratory Press. Sherrington R, Brynjolfsson J, Petursson H et al (1988): Localization of a susceptibility locus for schizophrenia on chromosome 5. Nature 336(6195): 164-167. Southern EM (1975): Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Moi Biol 98: 503-517. Spitzer RL, W iliams J B ( 1985): Structured Clinical Interview for DSM-ill Diagnoses-Personality Disorders. New York: New York Psychiatric Institute. Spitzer RL, Endicott Z, Robins E (1978): Research Diagnostic Criteria for a Selected Group of Functional Disorders. New York: New York State Psychiatric Institute. St. Clair D, Blackwood D, Muir W, et al (1989): No linkage of chromosome 5ql I-ql3 markers to schizophrenia in Scottish families. Nature 339: 305-307. Weeks DE, Brzustovic L, Squires-Wheeler E, et a! (1990): Report of a workshop on genetic linkage studies in schizophrenia. Schizophr Bull 16: 673-686. Williamson R, Bowcock AM, Kidd K, et al (1990): Report of the DNA committee and catalogues of cloned and mapped genes and DNA polymorphisms. Cytogenet Cell Genet 55: 457-478.
83
Evidence against Linkage of Schizophrenia to Chromosome 5ql 1-q 13 Markers in Systematically Ascertained Families Joachim Hallmayer,* Wolfgang Maier, Manfred Ackenheil, Martha A. Ertl, Sabine Schmidt, Jtirgen Minges, Dirk Lichtermann, and Dieter Wildenauer
Ten pedigrees systematically ascertained in Germany were tested for linkage to chromosome 5ql l-q13. In order to replicate the previous report by Sherrington et al (1988), families with a bipolar family member were omitted from the lod score calculations, all diagnoses were based upon Research Diagnostic Criteria, and four different models of the affection status were calculated, including the model for which Sherrington et al calculated the highest lod scores. None of the families investigated showed a positive lod score. Using multipoint linkage analyses, we were able to exclude the region for which a positive linkage has been reported.
Introduction Family, twin, and adoption studies have indicated that the etiology of schizophrenia is genetic, at least in part. In 1988, evidence that a gene causing schizophrenia is located on chromosome 5ql 1.2-q13.3 was reported based on a schizophrenic uncle-nephew pair with partial trisomy of 5q (Bassett et al 1988). Sherrington et al conducted linkage studies with DNA markers spanning the proximal portion of the trisomy including the 5q11.2 breakpoint in families from Iceland and Britain (Gilliam et al 1989). They reported positive lod scores between schizophrenia and the probes p105-153Ra (D5S39) and plO5-599Ha (D5S76). Subsequent linkage studies (Aschauer et al 1990; Crowe et al 1991; Detera-Wadleigh et ai 1989; Kennedy et al 1988; McGuffin et ai 1990; St. Clair ct al 1989) excluded linkage to schizophlenia using the same probes as Sherrington et al, but in different families. In the study of Sherdngton et al (1988), lod scores reached much higher values when models using ~ broader definition of the affection status were tested. The highest loci score~ were calculated when family members who suffered from a variety of psychiatric
From the Departmentof Psychiatry, University of Mainz, Germany (JH, WM, JM, DL); and Department of Neurochemistry, Psychiatric Hospt'tel. University of Munich.,Gemtaay (MA, MAE, SS, DW). *Current address: Departmelltof Genetics, Stanford University School of Medicine, Stanford, CA. Address reprint requests to Dr. Joachim H~|lmayer,Departmentof Genetics, StanfordUniversitySchool of Medicine, Stanford, CA 94305. Received September 18, 1990; revised May 28, 1991. © 1992 Society o! Biologi,~alPsycbi,atry
t~06-3223/92/$05.00
84
BIOL PSYCHIATRY 1992;31:83-94
J. Hallmayeret al
disorders were also considered as affected in the analysis. The affection status also included diseases for which no higher prevalence in the relatives of schizophrenic probands has been described so far. This result suggested that a genetic predisposition for a variety of psychiatric diseases is transmitted by a single dominant gene. A major difference between the results of Sherdngton et al and some studies rejecting linkage (e.g., Detera-Wadleigh et al 1989; St. Clair et al 1989) is that in the latter studies, families with a bipolar family member are included in the analysis but the) had been omitted in the initial study. It was therefore suggested by Gurling et al (1989) that inclusion of bipolar disorder in the definition of the affection status resulted in nonreplication of th~ positive linkage. Different diagnostic criteria may also have led to differences in the studies. Diagnoses of the Swedish kindred (Kennedy et al 1988) were based upon DSMIll criteria whereas Sherdngton et al applied Research Diagnostic Criteria (RDC). In previous studies, families characterized by a high number of affected relatives were selected for linkage studies; it is not evident that they were selected in a systematic manner. This fact~ together with the remoteness of the area where the most informative families were derived, make replication of their study very difficult (Weeks et al 1990). In the present study, all families with a minimum number of affected fatally members according to family history were included in the linkage study. All families of patients treated for schizophrenia at the Psychiatric University Hospital in Mainz and Munich were asked to participate. If, according to family history, at least one first-debree relative and another first-degree or second-degree relative showed symptoms of schizophrenia or schizoaffective disorder a personal psychiatric imerview was carded out and blood was drawn from all available family members. Our recruitment of families was based only on the number of affected family members diagnosed as schizophrenic or schizoaffective by family history. All families fulfilling the criteria mentioned above were recruited for the linkage analysis regardless of the final diagnoses. Linkage studies were carried out on these families with markers in the region studied by Sherrington et al (1988). In order to make our study comparable to the study by Sherrington et al, all families with a bipolar family member were omitted from the loci score calculations, diagnoses were made according to RDC, and four different models of the affection status were calculated, including the model for which Sherrington et al calculated the highest lod scores.
Material and Methods Ten families of patients suffering from chronic schizophrenia, with at least two relatives showing symptoms of schizophrenia or schizoaffective psychosis according to family history, were included in the study. In none of these families was a person diagnosed as having bipolar disorder. Pedigree charts of these 10 families arc shown in Figurc 1. Best estimate diagnoses (Leckman et al 1982) of probands and all participating relatives were made by review of two interviews conducted by specially trained cesearch assistants, by all available medical records, and by information given by rclatives. The types of interviews used were the Schedule for Affcctive Disorders and Schizophrenia-Lifetime Version (SADS-LA) (Fyer et al 1~85) and the Structured Clinical Interview for DSM111 diagnoses-Personality Disorders,; (Spitzer and Williams 1985). The best estimates of two raters were comparcd. If there was no agreement, the individual files were reviewed on a case-by-case basis and a consensus diagnosis was established. All diagnoses were based upon RDC (Spitzer et al 1978). Lymphocytes from patients and participating rclatives were immortalized and cultured.
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY 1992;31:83-94
85
Family I
S
S
S
S
Family i?.0
S
a
a
S
S
•
S
Family 3
~
S
S
S
S
S
Family 4
8
S
m
S
Family $
S
S
~
Family 6
i
m
a
m
m
i
i
S
S
S
S
•
S
m
Family 7
S
S
8
S
Family 8
i
Family 9
S
S
S
i
ean~, lo
$
$
m
$
$
El 0 unaffected I • sch~ i • scldzom%cfivedisorder
g Q mjor delmmive disorder Figure 1. Pedigrees with several affected family members by schizophrenia. Asterisk indicates family members from whom blood has been drawn and genoUipings have been carried out with the markers described in the te~t,
86
J. Hallmayer et al
BIOL PSYCHIATRY 1992;3! :83-94
Table I. Probes, Loci, and Polymorphisms on Chromosome 5 Typed in the German Pedigrees Allele Locus
Pro~
Enzyme
Sym
D5S76
p105-5~)Ha
TaqI
D5S6
M4
BamHI
D5S39
p105-153~a
MspI
D5S78
p 105-7981~b
Mspl
AI A2 A3 A! A2 A3 AI A2 AI A2
Size (kb)
Freq
17.0 14.0 10.0 11.0 9.6 7.6 8.7 5.8 2.3 1.8
0.32 O. 16 0.52 0.33 0.52 O. 15 0.33 0.67 O.57 0.43
Williamsonct al 1990.
DNA was extracted tram cell lines, and 5-10 Ftg were digested overnight with the appropriate restriction enzymes to reveal the restriction enzyme polymorphism (Table 1). The digested DNA was electrophoresed on 0.8% agarose gel for 10--16 hr. The DNA was denatured and transferred to a nylon filter according ~o the method described by Southern (1975). Probes were labeled with 32p-dATP by the random primer oligonucleotide method (Feinb~rg and Vogelstein 1983). After prehybridization, the filters were hybridized at 65°C under standard conditions (Sambrook et al 1989). Washed filters were exposed to a Kodak XAR-film at -70°C. Films were developed after 3 and 14 days. Genotype assignment was done by researchers who were blind to the affection status of the subjects. Four probes were used in the present study (Table I):p105-153Ra identifying the locus D5S39, M4 (D5S6), p105-599Ha (D5S76), and p105-798Rb (D5S78) (Kiddet al 1989; Williamson et al 1990). Sherrington et al (1988) reported positive Iod scores using the probes p105-153Ra (D5S39) and p105-599Ha (D5S76). M4 (D5S6) is located between these two probes (Figure 2) and p105-798Rb (D5S78) is 9 centimorgans distal from p105153Ra (D5S39). Schizophrenia was assumed in the analysis to have a dominant mode of inheritance with incomplete penetrance, as in the study by Sherrington et al. Estimation of the penetrance parameters was based upon a large family study carried out in the area of Maiaz (Maier et al, unpublished data 1991). Lod scores were calculated under four different models of the phenotype (Table 2). IVlodel I restricted the affection phenotype to schizophrenia and schizoaffective psychosis. The phenotype of nonaffected individuals
0.07 |_
, D5876
'
0.06 t D5S6
'
0.09 I D5S39
'
I D5S78
Figure 2. Map of chromosome 5 showing the rv:gion of the typed markers (Giuffra et al 1989). Distances a-e given in Morgans.
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY 1992;31:83-94
87
Table 2. Models Defined for Calculations of Lod Scores Model
Affection status
Model !
Schizophrenia Schizoaffective disorder Schizophrenia Schizoaffective disorder Schizophrenia Schizoaffective disorder Schizotypal personality Disorder Major depressive disorder Sch-~phrenia Schizoaffective disorder Schizotypal personality Bim31ar disorder Major depressive disordel, Minor depressive disorder Alcoholism Anxiety disorders
Model 2 Model 3
Model 4
Nonaffection status
Penetrance (%)
Unknown Known
55
Known
77
Known
85
was tre:~:ed as unknown. In this model, the unaffected do not ¢on~bute to the lod score. This means that neither a false-negative diagnosis nor incorrect estimation of the penetrance affect the calculation of the loci score (Goldin and Martinez 1989). In model 2, a penetrance of 55% was assumed for schizophrenia and schizoaffective disorder. Model 3 (assuming 77% penetrance) included additionally all cases of major affective disorders and schizotypal personality disorde~,s as affected. Anxiety disorders, minor affective disorders, and alcoholism as well as all cases of model 3 were included in model 4 (assuming 85% penetrance). Model 4 is the model Sherrington et al (1988) used in obtaining the highest positive lod scores. Based upon the age of onset of schizophrenia and affective disorders, seven agedependent liability classes were established, for which the penetrance is raised until the maximum value is reached (55% for model 2, 77% for model 3, and 86% for model 4). For iod score calculations, the programs MLINK and LINKMAP of the program package LINKAGE (Version 4.91) (Lathrop and Lalouel 1984; Lathrop et al 1985) were used. Multipoint analysis was carried out against a fixed map of the loci D5S76, D5S6, D5S39, and D5S78 (Giuffra et al 1989) (Figure 2). Two four-point linkage maps were calculated: (1) D5S76-D5S6-D5S39 and (2) D5S76-D5S6-D5S78. Results Altogether, diagnoses and genotypes were available from 138 family members (Figure l) of l0 families. Of TIhese, 16 were diagnosed as having schizophrenia and another I l as schizoaffective. Jtn ~ddition, 25 family members had a major depressive disorder. Panic disolder was found in one family member. Personality disorders were diagnosed in three of the participating individuals; none of them belonged to the schizotypa! type. In one family (family 10), major depressive disorder was found to be bilateral. In the study by Sherri~igton et al 1988, only families with a single source of the illness were collected.
88
BIOL PSYCHIATRY 1992;31:83-94
J. Hallmayc.ret al
As the data of markers in the disease pedigrees should be consistent with published data from the reference families (Gershon et al 1990), we calculated two muitipoint analyses between the markers with the distances given by (3iuffra et al (1989). D5S76D5S6-D5S39 resulted in a loglikelihood difference of 7.7 and D5S76-D5S6-D5S78 of 6.8 with the recombination fractions given by Giuffra et al. Two-point lod scores between schizophrenia and the tested markers are shown in Tables 3 and 4. All lod scores are negative for recombination fractions of 0% and 5% except for p105-798Rb (D5S78) in model 1. Tight linkage between M4' (D5S6) and schizophrenia can be excluded in models 3 and 4. One problem with calculating lod scores for schizophrenia is that the mode of transmission, and therefore the true penetrance, is urLknown. The unaffected status can also b~ difficult to evaluate. Therefore, the preferred method is to use affected cases only, excluding the possibility of false-negative cases as in model 1 (Ott 1990). For this model, two-point iod scores are - i. i0 at 0% recombination for D5S76 and - i.29 for D5S6 at 0% recombination. Using multipoint analyses, all loglikelihood differences are less than - 2 in the region between D5S76 and D5S39 in models 2, 3, and 4 (Figures 3 and 4). Tkerefore, the entire region between D5S76 and D5S39 can be excluded for linkage. In models 3 and 4 the whole region between D5S39 and D5S7~ can be also excluded. Altogether, the exclusion area in model 2 is about 20 centimorgans, in model 3 ~bout 30, and in model 4 about 40.
Discussion Our results are in concordance with the reports from several laboratories (Aschauer et al 1990; Crowe et al 1991; Detera-Wadleigh et al 1989; Kennedy et al 1988; McGuffin et ai 1990; St. Clair et al 1989). So far all studies have failed to replicate the pos!tive findings of Sherrington et al (1988). Besides pure chance, failure to replicate the positive results has been explained by genetic heterogeneity, differences in the population studied, differences in the definition of the affection status, and the ascertainment of families (Baron et al 1990; Gershon et al 1990; Owen et al 1990). Genetic heterogeneity means different mutant alleles give rise to the same phenotype and are segregating independently in populations° As pointed out by Risch (1990), however, to conclude genetie heterogeneity in the absence of a Mendelian inheritance pattern, linkage evidence to a given chromosomal region must be replicated by at least one additional laboratory and replicated evidence for linkage to a second chromosomal region must be demonstrated. As neither criterion has been met, the contradictory results could be due either to nonreplication or to genetic heterogeneity. If genetic heterogeneity is a~sumed, negative iod scores have to be interpreted with caution as a lod score of less than - 2 as criterion is only valid in the case of homogeneity. None of the families studied by our ~ 0~p ~s~',ted in positive lod scores that would have indic~ted heterogeneity. McGuffin et al (1990) carried out linkage analyses with the data by Sherrington et al (1988) together with the n~gative results published so far. They found no evidence for heterogeneity among families in the stcdies showing no evidence for linkage and concluded that true heterogeneity is unlikely to exist. All evidence for heterogeneity is derived from the one positive finding by Sherrington et al. Given the large number of families showing no linkage, it is obvious that the number of linked families is quite small (Generate! and Kidd !99!).
BIOL PSYCHIATRY 1992;31:83-94
L i n k a g e to C h r o m o s o m e M a r k e r s in S c h i z o p h r e n i a
89
T a b l e 3. T w o - p o i n t L o d s c o r e s b e t w e e n S c h i z o p h r e n i a a n d M a r k e r s f o r C h r o m s o m e 5q I 1-13 D5S76 Recombination fraction Family
0.0
0.05
0.1
0.20
0.30
0.40
7
- !.77 -0.64 0.00 0.0! -0.18 -0.01 0,00
-0.9! -0.47 0.00 0.01 -0.13 -0.01 0,00
-0.53 -0.35 0.00 0.00 -0.09 -0,01 0,00
-0.16 -0.17 0.00 0.00 -0.04 -0.01 0,00
-0.02 -0.07 0.00 0.00 -0.01 0.00 0,00
0.02 -0.02 0.00 0.00 0.00 0.00 0,00
8
0.00
0.00
0.00
0.00
0.00
0.00
9 10
0.00 - 0.20 - 2.78
0.00 - 0.16 - 1.67
0.00 - 0.12 - 1.09
0.00 - 0.07 - 0.45
0.00 - 0.03 - 0.14
0.00 - 0.01 - 0.01
-0.65 0.20 0.00 - 0.04 0.05 - 1.49 0.00 0.00 0.01 -0.04 - 1.96
-0.35 0.17 0.00 - 0.04 0.04 - 0.74 0.00 0.00 0.01 -0.03 - 0.93
-0.21 0.14 0.00 - 0.04 0.03 - 0.48 0.00 0,00 0.01 -0.02 - 0.56
-0.07 0.09 0.00 - 0.03 0.02 - 0.23 0.00 0.00 0.01 0.00 -0.22
- 0.02
0.04 0.00 - 0.02 0.01 - 0.09 0.00 0,00 0,01 0.00 - 0.08
- 0.01 0.01 0.00 - 0.01 0.00 - 0.02 0.00 (L00 t3,00 o.00 - qL02
!
- 0.45
2 3 4 5 6 7
-0.17 0.02 0.02 -0,21 - 0.02 -0.01
- 0.25 -0.13 0.02 0.01 -0,16 - 0.02 -0.01
- 0.14 -0.10 0.01 0.01 -0,12 - 0.02 -0,01
- 0.03 -0.05 0.01 0.00 -0,06 - 0.01 0.00
0.01 -0.02 0.00 0.00 - 0.03 -0.01 0.00 0.00 0.00 0,01 - 0,03
0.02 -0.01 0f~) 0.00 - 0.01 0.00 0 00 0.00 0.00 0,00 0,01
- 0.06
- 0.03 0.00 0.00 0.00 -0.01 0.00 0,00 0.00 0,00 - 0,01 -0,04
6
Sum D5S6 l 2 3 4 5 6 7 8 9 10 Sum D5839
8
0.00
0.00
0.00
0.00
9 10
- 0.02 0.03 -0.81
- 0.01 0.03 -0.53
- 0.01 0.02 -0.35
- 0.01 0.02 -0.13
I
- 0.30
2 3 4 5 6 7 8 9 10
0.00 0.02 - 0.06 -0,19 0.00 - 0,02 0.00 0,01 -0,21 - 0.75
- 0.23 0.00 0.02 - 0.05 -0,15 0,01 - 0,02 0.00 0,01 -0,~.7 - 0.57
- 0.17 0.00 0.01 - 0.04 -0,11 0,02 - 0,02 0.00 0,01 -0,13 - 0.43
- 0.10 0.00 0.01 - 0.02 -0,06 0,02 - 0,01 0,00 0,00 -0,07 - 0.23
Sum
D5S78
Sum
0.00 0.00 -0.01 -0.03 0.01 0,00 0.00 0.00 - 0,03 -0,11
Schizophrenia was treated as a dominant disease and the penetrance was assumed to be 55% (model 2).
90
J. H a l l m a y e r et al
BIOL PSYCHIATRY 1992;31:83-94
Table 4. Two-point Lod Scores between Schizophrenia and Chromosome 5 q l 1-13 Markers Recombination fraction Marker
Model
0.00
0.05
O. 10
0.20
0.30
0.40
D5S76
1 2 3 4 l 2 3 4
- 1, IO -2,78 -0.86 - 1.46 - 1,29 - 1,96 -3.47 - 3.29 - 0.09 -0.81 - 1.88 - 1.08 0,26 -0.75 - !.81 -2.62
- 0.62 - 1.67 -0.19 -0.70 -0.80 - 0.93 -2.23 -2.12 - 0.01 -0.53 - 1.25 -0.73 0.22 -0.57 - 1.39 -2.01
- 0.37 - 1.09 0,07 -0.31 -0.54 - 0,56 - 1.56 - 1.47 0.03 -0.35 -0.85 -0.48 O. 18 -0,43 - 1.05 - 1.50
- O. 12 -0.45 0.21 0,01 -0.25 - 0,22 -0,75 -0,68 0.05 -0.13 -0.37 -0.18 0.09 -0.23 -0.54 -0.75
- 0.02 -0.14 0.15 0.07 -0.10 - 0.08 -0.30 -0,26 0,04 -0,03 -0,13 -0.05 0.03 -0.11 -0.22 -0.30
O.O! -0.01 0.06 0,03 -0.02 - 0.02 -0.07 -0.05 0.02 0,01 -0.02 0.O0 0.00 -0.04 -0.05 -0.07
D5S6
D5S39
1
D5S78
2 3 4 I 2 3 4
Four different definitions of the affection status were calculated.
0 -1
l
-2 -3
mm
]J - 4 -5 m
-6 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Distance in Morgans Figure 3, Multipoint analyses o f schizophrenia against a fixed map o f DSS76-DSS6-D5S39. D 5 S 7 6 was set arbitrarily at 0,2 M on the map, The other markers were set according to genetic distances (Figure 2 and Giuffra et al 1989), Four different models of the affection status were calculated as defined in Table 2. Model ! (=) model 2 (n) model 3 (&); model 4 (A).
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY ! 992;31:83-94
91
0 -1
§
-2
|
-3
c 0
-4 -5
¢/) tO
C1
-6 0
0.1
0.2
¢n ~
t43
~t[
a
0.3
0.4
0.5
0.6
0.7
0.8
Distance in Morgans Figure 4. Multipointanalyses of schizophrenia against a fixed map of DSS76-D5S6-DSS78. D5S76 was set arbitrarily at 0.2 M on the map. The other markers were set according to genetic distances (Figure 2 and Giuffra et al 1989). Four different models of the affection status were calculated as defined in Table 2. Symbols same as in Figure 3.
One reason for the presence of a rare gene in the families collected by Sherrington et al may be the remote area from which the families were collected; five families are of Icelandic origin. Iceland is genetically isolated from the rest of the world (Cavalli-Sforza et al 1991) and the population genetically closest to Icelanders is that of Norwegians. Therefore, it may be that the families collected by our group differ genetically from these Icelandic pedigrees. On the other hand, two families from the United Kingdom (one from Wales) studied by Sherrington et al (1988) also gave positive lod scores whereas in six other families of South Wales origin no li~'~kagecould be detected between chromosome 5 markers and schizophrenia (McGuffin et al 1990). Given these results, a specific Icelandic "schizophrenia gene" seems unlikely. The greatest difficulty in carrying out linkage analysis in psychiatric disorders is defining the affected phenotype and estimating the parameters describing the genetic model. Recently it was shown that maximization of the lod score (Clerget-Darpoux et al 1990) calculating different models of the affection status leads to an increased probability of false-positive results. The highest lod scores in the study of Sherrington et al were found by including a great variety of psychiatric diseases. The penetrance was estimated by maximizing the lod scores with increasing penetrance values. Th;~s model is the same as model 4 in our study with the exception that the gene frequency in our model is much higher. The penetrance values as well as the gene frequencies were fixed before calculating the Iod scores. Lowering the gene frequency to the values used by Sherrington et al results in even lower lod scores (data not shown).
92
J. Hallmayeret al
BIOL PSYCHIATRY 1992;31:83-94
0 -1 =o
-2 -3
-7
-8
0
0.1
0.2
0.3
0.4
0.5
0.6
Distance in M0rgans Figure 5. Multipoint anlayses of schizophrenia against a fixed map of DSS76-D5S6-DSS39. The calculations include five families in which at least 1 family member was diagnosed as bipolar. D5S76 was set arbitrarily at 0.2 M on the map, The other markers were set according to genetic distances (Figure 2 and Giuffra et al 1989), Four different models of the affection status were calculated as defined in Table 2. Symbols same as in Figure 3,
The determination that a person is truly affected presents a major problem in psychiatry. Most psychiatric diseases have a variable age of onset, and milder form of psychopathology may be considered normal. The preferred method of detecting linkage is to carry out an analysis only on affected subjects, while considering that the unaffected have unknown phenotypes. This "conservative" procedure does not decrease the ability to detect or exclude linkage; however, the sample size needed to detect and exclude linkage is increased (Goldin and Martinez 1989). Two-point lod scores for this model (model 1) were negative for D5S76, D5S39, and D5S6 but they did not reach the significance level of less than - 2. Five more families have been collected in which the diagnostic evaluation showed that at least one family member was diagnosed as being bipolm-. Inclusion of these five families in the calculations resulted in significantly negative lod scores (Figure 5) also for model ! as well as for models 2, 3, and 4. Whether families with bipolar disorder should be included in linkage studies of schizophrenia is controversial. The studies of Detera-Wadleigh et al (1989) and St. Clair et al (1989) both collected families with bipolar disorder, in a recent controlled family study it was shown that manic and schizophrenic syndromes were transmitted independently in families. Major depression, however, turned out to be an indic~or for a higher susceptibility tbr schizophrenia and bipolar disorder (Maier et al unpublished data 1991). Given these results, it can be speculated that two different genes--one a ,,usceptibility gene causing schizophrenic syndromes, one causing mania--both result in a higher vul-
Linkage to Chromosome Markers in Schizophrenia
BIOL PSYCHIATRY 1992;31:83-94
93
nerability to unipolar depression. Inclusion of families with bipolar disorder would therefore result in two different genes causing unipolar depression. The detection of linkage for the broader definition of the phenotype would be difficult. On the other hand, restriction of the phenotype to a narrow definition of the phenotype should not decrease the chance of detecting linkage. Though we have applied a methodology similar to that used by Sherrington et al (1988), we were unable to find any evidence for dominant susceptibility gene for schizophrenia on chromosome 5q I l-q13 in our families from Germany. We wish to thank Prof. Dr. Mundt, Dr. Schleuning, and Dr. Minelli for their help in collecting and diagnosing the pedigrees. We also thank F. Schmidt, S. Scl~,wab, and U. Schoppe for preparation of probes and enzyme digestions. We are grateful to J. Mountain for he~ critical comments. All probes were received through ATCC. Dr. Hallmayer is supported by a fellowship of the Deutsche Forschungsgemeinschaft. This study was supported by the Deutsche Forschungsgemeinschafi grant numbers Wi 453/7-1 and Ma ! 142tl-3.
References Aschauer HN, Aschauer-Treiber G, Isenberg KE, et al (1990): No evidence for linkage between chromosome 5 markers and schizophrenia. Hum Hered 40:109-115. Baron M, Endicott J, Ott J (1990): Genetic linkage in mental illness. Limitations and prospects. Br J Psychiatry 157: 645--655. Bassett A J, Mc Gillivray BC, Jones BD, Pantzar JT (1988): Partial trisomy 5 cosegregating with schizophrenia. Lancet 9: 799-801. Cavalli-Sforza LL, Piazza A, Menozzi P (1991). Histmy and Geography of Human Genes. Princeton University Press. Clerget-Darpoux F, Babron M-C, and Bonaiti-Pellie C (1990): Assessing the effect of multiple linkage tests in complex diseases. Genet Epidemiol 7: 245-253. Crowe RR, Black DW, Wesner R, Andreasen NA, Cookman A, Roby J (1991): Lack of linkage to chromosome 5ql I-q13 markers in six schizophrenia pedigrees. Arch Gen Psychiatry. 48: 357-361. Detera-Wadleigh SD, Goldin LR, Sherrington R, et al (1989). Exclusion of linkage to 5ql 1-13 in families with schizophrenia and other psychiatric 0isorders. Nature 340: 391-393. Feinberg AP, Vogelstein B (1983): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1326-1333. Fyer AZ, Endicott Z, Mannuzza S, Klein DF (1985): Schedule for Affective Disorders and Schizophrenia, Lifetime Version (SADS.LA). New York: New York Psychiatric Institute. Gelernter J, Kidd KK (1991): The current status of linkage studies in schizophrenia. In McHugh PR, McKusick VA (eds.), Genes, Brain, and Behavior (New York: Raven Press, pp 137-152. Gershon ES, Martinez M, Goldin LR, Gejman PV (1990): Genetic mapping of common diseases: The challenges to manic-depressive illness and schizophrenia. Trends Genet 6: 282-287. Gilliam TC, Freimer NB, Kaufmann CA, et al (1989): Deletion map-"ag of DNA markers to region of chromosome 5 that cosegregates with schizophrenia. Genom -s 5: 940-944.
a
Giuffra LA, Kennedy JL, Pakstis AJ, et al (1989): A linkage group of ,., litarkers spanning >100 cM on chromosome 5 based on non-CEPH families. Cytogenet Cell Genet 51: 1004. Goldin LL, Martinez MM (1989): The detection of linkage and heterogeneity in nuclear families when unaffected individuals are considered unknown. Muhipoint Mapping and Linkage Based
94
BIOL PSYCHIATRY 1992;31:83-94
J. Hallmayer et al
Upon Affected Pedigree Members: Genetic Analysis Workshop 6. New York: Alan R. Liss, pp 195-200. Gurling HMD, Sherrington RP, Brynjolfsson J, et al (1989): Recent and future molecular genetic research into schizophrenia. Schizophr Bull 15: 373-382. Kennedy JL, Giuffra LA, Moises HW et al (1988): Evidence against linkage of schizophrenia to markers on chromosome 5 in a northern Swedish pedigree. Nature 336: 167-170. Kidd KK, Bowcock AM, Schmidke J, et al (1989): Report of the DNA committee and catalogs of cloned and mapped genes and DNA polymorphisms. Cytogenet Cell Genet 51: 622-947. Lathrop GM, Lalouel JM (1984): Easy calculations of lod scores and genetic risk on small comput.ers. Am J Hum Genet 36:460-465. Latlu'op GM, Lalouel JM, Julier C, Ott J (1985): Multilocus linkage analysis in humans: Detection of linkage and estimation of recombina'~ion. Am J Hum Genet 37: 482-498. Leckman, JF, Sholomskas D, Thompson D, Belanger A, Weissmann MM (1982): Best estimate of lifetime psychiatric diagnosis. Arch Gen Psychiatry 39: 879-883. McGuffin P, Sargeant M, Hetti G, Tidmarsh S, Whatley S, Marchbranks RM (1990): Exclusion of a schizophrenia susceptibility gene from the chromosome 5q I l-q 13 region: New data and a reanalysis of previous reports. Am J Hum Genet 47: 524-535. Ott J (1990): Genetic linkage and complex diseases: A comment. Genet Epidemiol 7: 35-36. Owen M, Crauford D, St Clair D (1990): Localization of a susceptibility locus for schizophrenia on chromosome 5. Br J Psychiatry 157: 123-127. Risch N (1990): Genetic linkage and complex diseases, with special reference t(~ psychiatric disorders. Genet Epidemiol 7: 3-16. Sambrook J, Fritsch EF, Maniatis T (1989): Molecular Cloning. A Laboratory Manual (ed 2). Cold Spring Harbor, NY: Laboratory Press. Sherrington R, Brynjolfsson J, Petursson H et al (1988): Localization of a susceptibility locus for schizophrenia on chromosome 5. Nature 336(6195): 164-167. Southern EM (1975): Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Moi Biol 98: 503-517. Spitzer RL, W iliams J B ( 1985): Structured Clinical Interview for DSM-ill Diagnoses-Personality Disorders. New York: New York Psychiatric Institute. Spitzer RL, Endicott Z, Robins E (1978): Research Diagnostic Criteria for a Selected Group of Functional Disorders. New York: New York State Psychiatric Institute. St. Clair D, Blackwood D, Muir W, et al (1989): No linkage of chromosome 5ql I-ql3 markers to schizophrenia in Scottish families. Nature 339: 305-307. Weeks DE, Brzustovic L, Squires-Wheeler E, et a! (1990): Report of a workshop on genetic linkage studies in schizophrenia. Schizophr Bull 16: 673-686. Williamson R, Bowcock AM, Kidd K, et al (1990): Report of the DNA committee and catalogues of cloned and mapped genes and DNA polymorphisms. Cytogenet Cell Genet 55: 457-478.