linkage studies of bipolar disorder in the region of the darier's disease gene on chromosome...

American Journal of Medical Genetics (Neuropsychiatric Genetics) 60:94-102 (1995)

Linkage Studies of Bipolar Disorder in the Region of the Darier’s Disease Gene on Chromosome 12q23-24.1

Elisabeth Dawson, Elizabeth Parfitt, Queta Roberts, Jo Daniels, Lionel Lim, Pak Sham, Markus Nothen, Peter Propping, Mario Lanczik, Wolfgang Maier, Ulrike Reuner, Jean Weissenbach, Michael Gill, John Powell, Peter McGuffin, Mike Owen, and Nick Craddock Department of Psychological Medicine (E.P., Q.R., J.D., P.M., M.O., N.C.), Department of Medical Genetics (E.P., Q.R., J.D., M.O.), University of Wales College of Medicine, Heath Park, Cardifi Departments of Neuroscience and Psycho- logical Medicine, Institute of Psychiatry (E.D., L.L., P.S., M.G., J.P.), London, United Kingdom; Institute of Human Genetics, University of Bonn (M.N., P.P.), Bonn, Psychiatric Clinic, University of Wiirzburg (M.L.), Wiirzburg, Psychi- atric Clinic, University of Mainz (W.M.), Mainz, Psychiatric Clinic, Medical Academy (U.R.), Dresden, Germany; GenB thon (J . W.), Evry, France; Department of Psychological Medicine, National University of Singapore (L.L.), Singapore; and Department of Psychiatry, Washington University School of Medicine, Jewish Hospital (N.C.), S t Louis, Missouri

We have recently described a family in which there is cosegregation of major affective disorder with Darier’s disease and have mapped this autosomal dominant skin disorder to 12q23-q24.1. This has provided an interesting candidate region for genetic studies of bipolar disorder. We have studied the segregation of seven markers spanning the Darier’s disease locus in 45 bipolar disorder pedigrees and found modest evidence in support of linkage under heterogeneity for 5 of these markers. Nonparametric analyses were suggestive of linkage with a marker at the gene encoding a secretory form of phospholipase A2. Our sample has relatively low power to detect linkage under heterogeneity and independent researchers should examine markers from this region in further samples of bipolar pedigrees. 0 1995 Wiley-Liss, Inc.

KEY WORDS: affective disorder, candidate gene, DAR locus, PLA2

INTRODUCTION There is compelling evidence from family, twin and

adoption studies for the existence of important genetic factors determining susceptibility to bipolar affective

Received for publication January 3, 1994; revision received March 4, 1994.

Address reprint requests to Drs. Nick Craddock and Mike Owen, Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kmgdom.

0 1995 Wiley-Liss, Inc.

disorder [Craddock and McGuffin, 19931. However, the mode of inheritance is unknown. Simple mendelian transmission may occur in some families but cannot explain the majority of cases [Sham et al., 19921, which probably reflect the action of several genes together with environmental factors. With the advent of poly- morphic DNA markers, linkage and association studies have become potentially useful methods for genetic analysis of affective illness, but as yet, no consistent findings have emerged.

An important problem in molecular genetic studies is deciding where in the genome to look for susceptibility loci. A random or systematic “genome search” may ulti- mately prove necessary but the more focused approach of examining “candidate” regions may be particularly efficient. A region can be considered a “candidate” if there exists some a priori evidence that a susceptibility locus may be located within it. This evidence may come from: (1) knowledge that a gene encoding an enzyme or structural protein thought to be important in the pathogenesis of affective disorder lies within the region [e.g., Leboyer et al., 19901, (2) the discovery of cytogenetic abnormalities associated with bipolar disorder [Craddock and Owen, 19941, and (3) the finding of cosegregation within families of bipolar disorder and a monogenic disorder. Such an observed cosegregation formed the rationale for this study.

If bipolar disorder and a disease (or trait) determined by a single gene are associated within families, there are a number of possible causes for this: (1) the two disorders are different manifestations of the same genetic defect (pleiotropy), (2) they are caused by defects in genes close enough together on the same chromosome to display linkage, and (3) they could be part of a contiguous deletion syndrome, the chromosomal deletion being too small to be detectable by routine cytogenetic methods.

Bipolar Linkage Studies at the Darier’s Locus 95

addition, Korner et al. [1993] have reported a family in which affective disorder and Hailey-Hailey disease are associated. Hailey-Hailey, like Darier’s disease, shows autosomal dominant transmission. Moreover, there is an overlap of both morphological and histological features between these conditions and it has been suggested that they might be caused by different allelic mutations a t a single locus [Buxton, 19931.

The recent localization of the Darier’s disease gene (DAR) to chromosome 12q23-24.1 [Craddock et al., 1993; Bashir e t al., 1993; Parfitt et al., 19941 has thus provided an interesting region for linkage studies of major affective disorder.

MATERIALS AND METHODS Pedigrees

Pedigrees for use in linkage studies of bipolar disorder were collected by three collaborating groups: Cardiff (C), London (L) and Bonn (B). The groups are members of the larger collaborative European Science Foundation (ESF) Network on the Neurobiology of Mental Illness and followed the methodology adopted by the ESF [Leboyer and McGuffin, 19911. Available family members were interviewed using SADS-L [En- dicott and Spitzer, 19781 modified to provide information for DSMIII-R diagnoses. When available, hospital records were obtained and when necessary, information from additional informants was collected. Life- time, best-estimate psychiatric diagnoses according to DSMIII-R criteria were made upon the basis of all available data. The 45 pedigrees included in the current report were selected from a total of 66 pedigrees collected by the collaborating groups on the basis of the following criteria: (1) a sampled adult proband with DSMIII-R bipolar disorder [MA, 19873, (2) a t least one sampled sibling or second degree relative with DSMIII- R bipolar disorder or schizoaffective disorder (bipolar) or bipolar disorder, NOS, or recurrent major depression, (3) both parents of potentially informative sibships did not have disorders listed in (2) above. The purpose of criterion (3) was to exclude uninformative matings. No specific attempt was made to exclude bi- lineal pedigrees. Selection was made prior to genotyping. In 38 pedigrees a t least two members had DSMIII- R bipolar disorder. Table I provides information about the families included in the study.

Pedigree 324 in which cosegregation of Darier’s disease and major affective disorder occurs [Craddock et al., 19941 is not included in the set of pedigrees presented in the current report.

Genotyping The Darier’s disease gene has been

localized to the region around D12S84 and in our set of five Darier pedigrees (which includes pedigree 324) we observed no recombinants between the disease and this marker [Craddock et al., 19931. In our study of bipolar disorder, we typed D12S84 together with six neigh- bouring markers in 223 individuals from the 45 pedigrees. The order and sex averaged distance between the markers are as follows: cen-DlBSlOl-( llcM)-D12S78-

Markers used.

(5cM)-DlZS84-( 7~M)-D12S79-( llcM)-D12S76-(8cM)-

Craddock et al. [1994] recently reported a British family (pedigree 324) in which there is intrafamilial association of major affective disorder with Darier’s disease (keratosis follicularis) and in which the occurrence of affective disorder does not appear to be the result of social disability or other nonspecific factors.

Darier’s disease is an autosomal dominant skin disease with a prevalence of 1 in 55,000 in the United Kingdom and is characterized by abnormal adhesion between keratinocytes resulting in warty brown pap- ules on the trunk and flexures [Burge and Wilkinson, 19921. Darier’s disease seems to be associated with mental retardation in some families [Burge and Wilkinson, 1992; Burge, 19931 and also with an increased prevalence of epilepsy [Burge and Wilkinson, 1992; Burge, 19931. There have been a few, mostly anecdotal, reports of depression and suicidal ideation in patients with Darier’s disease [Denicoff e t al., 1990; Medansky and Woloshin, 1961; Peck et al., 1976; Svendsen et al., 19591 and of the cooccurrence of bipolar disorder [Clark et al., 1986; Milton et al., 19901. However, in the only large systematic study of Darier’s disease to date, affective disorder was no more common than expected from population rates in the 163 cases studied [Burge and Wilkinson, 1992; Burge and Crad- dock, unpublished observations]. A strong association between Darier’s disease and major affective disorder is, therefore, unlikely.

In pedigree 324 [Craddock et al., 19941 there is cooccurrence of major affective disorder and Darier’s disease in five members. One of the affected individuals has bipolar disorder, the other four have major depression. In our report [Craddock et al., 19941 we described absence of both disorders in five members. However, the youngest member of the sibship has recently demonstrated the features of Darier’s disease but has not yet had an affective episode. Pedigree 324 with the current diagnostic status of all individuals is shown in Figure 1. The pedigree is consistent with genetic linkage between the Darier gene and a major autosomal dominant susceptibility locus for major affective disorder and, using an “affected only” lod score analysis using the genetic parameters in Table I1 for the broad (B) diagnostic model, provides a maximum lod score of 0.91 a t zero recombination in support of such linkage. There has been one previous report of cosegregation between severe psychiatric disorder including affective disorder and Darier’s disease [Getzler and Flint, 19661 although the psychiatric phenotypes were poorly described. In

T*’ Fig. 1. Pedigree 324. Filled circles and squares indicate major

affective disorder. D indicates Darier’s disease.

96 Dawson et al.

TABLE I. Characteristics of the 45 Families Included in the Current Study

Pedigree set Cardiff Bonn London Total No. families No. DNA samples No. affected subjects (N)" No. affected subjects (N)

with DNA available Male:femaleb ratio of affected

subjects (N) No. affected subjects (B)" No. affected subjects (B)

with DNA available Ma1e:female ratio of

affected subjects (B)

22 98 51 47

0.89:1

62 56

0.72:l

16 76 31 30

1:l

42 40

0.91:l

7 45 38 212 16 98 15 92

0.67:1 0.85:l

23 127 21 117

0.53:1 0.74:1

"N denotes the narrow and B the broad diagnostic category (refer to text for definitions). bThe differences in ma1e:female ratios of affected subjects were not statistically significant.

AF'M294ze9-tel [Weissenbach et al., unpublished]. The marker a t PLAB has not been integrated into the GQnQthon map using CEPH pedigrees. Dawson et al. [1993], using 23 pedigrees multiply affected by schizophrenia, observed no recombination between PLA2 and S76. In our bipolar dataset, PLAB is linked to S76 with a maximum likelihood estimate of distance of 2.4cM. The gene has been physically mapped, using in situ hybridization to 12q23-24.1 [Seilhamer et al., 19891, and using somatic cell hybrids to a position telomeric to D12S76 (Kolble, personal communication).

Methodology. High molecular weight genomic DNA was extracted from whole blood or transformed lymphoblasts according to routine procedures. Individ- uals were genotyped using the polymerase chain reac- tion (PCR) [Saiki e t al., 19851. The markers were visu- alized following end-labelling of one of the PCR primers with 33P-dATP or 32P-dATP followed by electrophoresis of the PCR products on denaturing polyacrylamide gels and autoradiography. Autoradiographs were scored and then read independently by a coworker.

Statistical Analyses Two-point linkage analyses

were performed between the putative major locus for affective disorder and each of the seven chromosome 12 markers using MLINK from the LINKAGE (version 5.1) package of programs [Lathrop et al., 19841. The affective illness observed in pedigree 324 follows an auto- soma1 dominant pattern of inheritance. Therefore, in this study, we assumed an autosomal dominant mode of transmission for the putative affective disorder susceptibility locus. We used two diagnostic models: narrow (N) in which only subjects with DSMIII-R bipolar disorder were considered to be affected, and broad (B) in which subjects with any of the following DSMIII-R diagnoses were considered to be affected: (1) bipolar disorder or (2) schizoaffective disorder with a t least one episode of schizomania, or (3) bipolar disorder, not otherwise specified, or (4) recurrent major depression. I t should be noted that the broad diagnostic model re- flects more closely the spectrum of affective disorder in pedigree 324 than does the narrow model. In our analyses all individuals who were not classified as affected were coded as phenotype unknown. The genetic para-

Lod score analysis.

meters used in the analyses are shown in Table 11. These parameters reflect the following assumptions: (1) a population lifetime prevalence of 1% for the N diagnostic category and 6% for the B category (Weissman et al., 1988), (2) a penetrance of 50% for both the disease gene heterozygote and homozygote state, and (3) phenocopy rates of 10% for the N category and 50% for the B category. In our present state of knowledge of the genetics of bipolar disorder, the choice of genetic parameters is to some extent arbitrary. Our choice of gene frequencies and phenocopy rates represent a "best guess" of the true values and the resulting analysis should be robust to their misspecification [Ott, 19911. We treated unaffected individuals as having unknown phenotype in order to minimize errors due to misspecification of the penetrance vector. This is achieved a t the expense of reduction in potential linkage information. However, we believe that the linkage information lost is of low quality which could have introduced considerable random "noise" into the analysis. Analyses were performed for pedigrees from each centre separately and for the combined set of pedigrees. Marker allele frequencies used in the lod score and nonparametric analyses were estimated from approximately 170 unrelated individuals within these pedigrees and other pedigrees typed by our laboratories for these markers.

Heterogeneity tests were performed using the A Test as implemented in the HOMOG program [Ott, 19911 and the Liang test [Liang, 19941. Both tests are based on a mixture model which assumes that a proportion a of the pedigrees are linked (i.e., recombination fraction, 0 < 0.5), while the others are unlinked (0 = 0.5). The

TABLE 11. The Genetic Parameters Used in the Lod Score Analysis

Diagnostic model fdda fdDa fDD" g" Narrow (Nib 0.001 0.5 0.5 0.009 Broad (B)b 0.032 0.5 0.5 0.03

afdd, fd,) and fuu are the penetrances of the dd, dD and DD genotypes, respectively, where D represents the putative disease susceptibility allele and d the wild type allele. q is the population frequency of allele D. bSee text for definitions of the N and B diagnostic categories.


which were assigned equal allele frequencies such that the sum of the frequencies of all four groups was unity. This method of recoding is conservative in that it will act in the direction of reducing evidence of ibd sharing in sib pairs in which missing marker information is estimated by the program.

We also analysed the data using the APM method of Weeks and Lange [19881. This uses identity by state (ibs) scores to analyse marker information only from pairs of affected relatives. Because it is more striking for dis- tantly affected relatives to share a rare marker allele than a common marker allele, the test statistic of Weeks and Lange [19881 includes a weighting factor, F(p), based on allele frequency. The authors proposed three possible weighting functions: F(p) = 1, F(p) = l/SQR(p) and F(p) = l/p (where SQR indicates square root) and suggested that the function F(p) = l/SQR(p) probably offers the best compromise between incorporating information about allele frequencies whilst gen- erating a normally distributed test statistic. However, it should be noted that if the marker allele frequencies are uncertain, as is often the case with highly poly- morphic markers, then weighting function F(p) = 1 is the most robust because it does not depend on allele frequency.

RESULTS Lod Score Analysis

The results of the two-point linkage analyses between bipolar disorder and each of the seven markers is shown in Table I11 for the narrow (N) diagnostic model and Table IV for the broad (B) model. The results of the heterogeneity tests are shown in Table V.

Power Studies Using the broad diagnostic model and under the

assumption of linkage homogeneity, our sample has a power (defined as the probability of obtaining a maximum lod score of greater than or equal to 3) of 0.97 to detect linkage at 8 = 0.00 and 0.70 at 8 = 0.05. The power is greatly reduced under the assumption of linkage heterogeneity. When only half the pedigrees were assumed to be linked, the power drops to 0.13 at 8 = 0.00 and 0.02 at 8 = 0.05.

ESPA Analysis The results of the sib pair analyses for the broad di-

agnostic category (B) are shown in Table VI. Results for the N category are not shown but demonstrate a trend towards increased sharing of alleles by affected sib pairs for markers S78, S84, S79, S76 and PLA2. The trend does not reach significance at the P < 0.05 level for any of these markers.

APM Analysis

b) Affected pedigree member (APM) analysis.

The results of the APM analyses for the broad diagnostic category (B) are shown in Table VII.

DISCUSSION The pattern of affective illness in pedigree 324 is sug-

gestive of autosomal dominant transmission and, thus, provides a very specific and testable hypothesis: i.e.,

null hypothesis is the same in both tests, being specified by setting (Y = 0. The alternative hypothesis in both tests is obtained by relaxing this constraint, i.e., by let- ting (Y > 0. The A test uses the conventional likelihood ratio statistic of the two hypotheses, but because the 8 parameter for the linked pedigrees is meaningless under the null hypothesis (i.e., (Y = 0), the regularity conditions necessary for the statistic to asymptotically approach a chi-squared distribution are violated. Consequently, the asymptotic distribution of the A statistic is not well established [Faraway, 19921. Moti- vated by this, Liang [1994] devised a new statistic to compare the two hypotheses, defined as the sum of the antilog,, of the pedigree lod scores a t the overall maximum likelihood estimate of 8 (assuming homogeneity of 8) minus the number of pedigrees. He showed that this statistic is asymptotically distributed as a 5050 mixture of a point mass a t 0 and a chi-squared distribution with one degree of freedom. Preliminary power studies performed by Liang [ 19941 suggest that the Liang test is a t least as powerful as the conventional likelihood ratio test, when null distributions are obtained by simulation.

The power of the sample to detect linkage using the broad diagnostic model was examined by simulation studies using the SIMLINK program [Ploughman and Boehnke, 19891. We used the same genetic parameters for the simulation as were used in the lod score analysis (see Table 11). Two thou- sand replicates of the pedigree set were made under the assumption of linkage homogeneity and 1,000 under the assumption that only half the pedigrees were linked to the marker locus. The simulated marker had four equally common alleles (corresponding to a marker polymorphism information content of approximately 0.7). A more detailed description of simulation studies using a slightly larger dataset incorporating most of the present families is given by Lim et al. [1994].

Because the mode of inheritance in bipolar disorder is unknown, we also examined the data using two methods of analysis which do not rely upon assumptions about the mode of transmission or upon estimates of genetic parameters.

We analysed the data using the ESPA program [Sandkuyl, 19891. This program analyses identity by descent (ibd) scores in pairs of affected siblings and makes use of marker data from other members of the pedigree to recon- struct, where possible, the marker genotypes of any missing members of the sib pair-parent quartet. Where this is not possible, a likelihood method is used to estimate the probability distribution of missing genotypes. Multiple affected sib pairs within one family are treated as being independent. A one-tailed chi-square test is used to detect significant departures ofibd score from expectation under the null hypothesis of no linkage. The program in its current form accepts a maximum of 5 alleles per marker. All markers used in this study had at least 6 alleles. Therefore, alleles were recoded to 4 per marker. Any allele with a frequency of over 0.25 had its identity and frequency preserved in the recoding. Alleles with frequencies less than 0.25 were recoded into one of the other allele categories

Power of the sample.

Nonparametric analyses.

a) Extended sib pair analysis (ESPA).

98 Dawson et al.

TABLE 111. Results of Two-Point Linkage Analysis Between Affective Disorder and Seven Markers for the Narrow (N) Definition of Affected Status

Marker Dataset 0.00 0.01 0.05 0.10 0.20 0.30 0.40

SlOl

S78

C" L B T C L B T

S84 C L B T

s79 C L B T

S76 C L B T

PLA2 C L B T

AFM294ze9 C L B T

-4.83 0.28

-2.31 -6.86 -1.93 -0.31 -1.94 -4.18 -2.27

0.27 -6.08 -8.08 -1.62 -0.70 -4.86 - 7.18

0.60 0.07

-5.33 -4.66 -0.55

0.70 -5.08 -4.93 -2.89 -1.09 -5.46 -9.44

-4.04 0.27

- 1.86 -5.63 -1.25 -0.16 -1.47 -2.88 -1.57

0.26 -4.88 -6.19 -1.06 -0.54 -3.87 -5.47

0.60 0.08

-4.38 -3.70 -0.08

0.70 -4.15 -3.53 -2.35 -0.81 -4.43 -7.59

-2.53 0.25

-1.06 -3.34 -0.13

0.10 -0.71 -0.74 -0.32

0.23 -2.85 -2.94 -0.02 -0.22 -2.24 -2.48

0.59 0.12

-2.69 -1.98

0.68 0.65

-2.49 -1.16 -1.28 -0.37 -2.58 -4.23

-1.59 0.20

-0.61 -2.00

0.35 0.19

-0.33 0.21 0.27 0.18

-1.75 - 1.30

0.46 -0.07 -1.37 -0.98

0.54 0.15

-1.73 -1.04

0.92 0.59

-1.57 -0.06 -0.66 -0.14 -1.58 -2.38

-0.64 0.12

-0.21 -0.73

0.52 0.18

-0.07 0.63 0.53 0.11

-0.74 -0.10

0.61 0.03

-0.57 0.07 0.37 0.14

-0.75 -0.24

0.81 0.43

-0.68 0.56

-0.15 0.02

-0.63 -0.76

-0.22 0.06

-0.06 -0.22

0.33 0.10 0.00 0.43 0.36 0.05

0.14 0.39 0.03

-0.21 0.21 0.19 0.09

-0.29 -0.01

0.46 0.25

-0.26 0.45

-0.01 0.04

-0.22 -0.19

-0.27

-0.04 0.02

-0.02 -0.04

0.10 0.03 0.00 0.13 0.11 0.01

-0.06 0.06 0.12 0.01

-0.05 0.08 0.05 0.03

-0.07 0.01 0.13 0.08

-0.06 0.15 0.01 0.02

-0.06 -0.03

"Results are shown for Cardiff (C), London (L) and Bonn (B) sets of pedigrees as well as for total (T) dataset.

the existence of a major susceptibility locus for severe affective disorder located in the region of the Darier's disease gene, which results in an autosomal dominant mode of transmission. The lod score method is, therefore, most appropriate for the analysis of this dataset because it is able to use information about the pre- sumed mode of inheritance.

The results of lod score analysis using a narrow definition of affected suggests that we can reject the hypothesis of tight linkage under homogeneity with the genetic model used for all the markers examined. The lod scores are less than -2 at 8 = 0 for each marker. With the broader diagnostic model, tight linkage under homogeneity can be rejected for SlOl and AFM294ze9. However, for the intervening markers lod scores are greater than -2 at zero recombination and tight linkage under homogeneity cannot be rejected.

For markers S78, S84, S79, S76, and PLA2 there is some evidence in favour of linkage, with small positive maximum lod scores for both narrow and broad diagnostic models. The ESPA analysis also provides evidence in favour of linkage with increased sharing of marker alleles ibd above chance expectation for all these markers. This increased sharing approaches statistical significance only for S78 (P < 0.053), S79 (P < 0.061) and PLA2 (P < 0.078). The APM method of analysis shows some evidence for increased sharing of alleles ibs for these markers but only approaches statistical significance for PLA2 using the F(p) = l/p weight-

ing (P < 0.05). However, using the F(p) = 1 weighting function) which is the function most robust to misspecification of marker allele frequencies) there is no evidence for increased sharing of marker alleles ibs.

The APM method shows significantly increased sharing of marker alleles ibs in affected pedigree members for marker SlOl (P < 0.005 for weighting function F(p) = l/SQR(p)). This is inconsistent with results from both the lod score and ESPA methods which, because they use ibd information) make more efficient use of data from nuclear and extended families and are more robust to misspecification of allele frequencies [Bishop and Williamson, 1990; Babron et al., 19931. This APM result is almost certainly spurious and highlights the importance of using ibd methods where possible.

The marker a t PLAB shows the strongest evidence in favour of linkage. The maximum lod score is 0.60 at 8 = 0.22 with PLAB for the narrow diagnostic model and 0.91 at 8 = 0.149 with PLA2 for the broad model. Inspection of the lod scores according to investigating centre (Tables I11 and IV) shows that families from Cardiff and London contribute positive lod scores whereas those from Bonn contribute negative lod scores to the total. Within each centre, there are both families contributing positive and negative lod scores. The A test shows a small increase in maximum lod score when heterogeneity is allowed but does not significantly favour the hypothesis of linkage under heterogeneity against that of linkage under homogeneity.

Bipolar Linkage Studies at the Darier's Locus

TABLE IV. Results of Two-Point Lod Score Analysis Between Affective Disorder and Each of Seven Markers for the Broad (B) Definition of Affected Status

99

0

Marker Dataset 0.00 0.01 0.05 0.10 0.20 0.30 0.40

SlOl C" L B T

S78 C L B T

S84 C L B T

s79 C L B T

S76 C L B T

PLAB C L B T

AFM294ze9 C L B T

- 1.96 0.22

-1.02 -2.76

0.26 -0.43 -0.05 -0.22

0.50 0.13

-1.61 -0.98

1.13 -1.57 - 1.43 -1.87

1.04 -0.68 -1.82 - 1.46

1.65 0.14

-1.51 -0.02 -0.66 -0.58 -2.11 -3.35

- 1.81

-0.93 -2.51

-0.37 -0.04 -0.08

0.55 0.14

0.23

0.33

- 1.50 -0.81

1.18 - 1.45 -1.32 - 1.59

1.04 - 0.62 -1.69 - 1.27

1.65 0.15

0.13 - 1.67

-0.58 -0.50 -1.96 -3.04

- 1.32

-0.67 -1.75

0.53 -0.20

0.01 0.34 0.67 0.17

0.24

-1.14 -0.30

- 1.05 -0.95 -0.72

1.01 -0.41 -1.26 -0.66

1.61 0.20

-1.25 0.56

-0.33 -0.29 - 1.44 -2.06

1.28

-0.98 0.23

-0.37 -1.12

0.62 -0.08

0.06 0.60 0.71 0.17

0.08 1.25

-0.71 -0.61 -0.07

0.92 -0.23 -0.88 -0.19

1.46 0.22

-0.84 0.84

-0.13 -0.14 -0.97 -1.24

-0.80

-0.38 0.16

-0.21 -0.43

0.54 0.02 0.07 0.63 0.58 0.13

-0.36 0.35 0.94

-0.31 -0.23

0.40 0.65

-0.06 -0.38

0.21 1.02 0.19

-0.36 0.85 0.04 0.00

-0.42 -0.38

-0.13 -0.03 0.08 0.02

-0.08 -0.01 -0.13 -0.02

0.31 0.09 0.03 0.01 0.04 0.01 0.38 0.11 0.32 0.09 0.07 0.02

-0.13 -0.02 0.26 0.09 0.51 0.14

-0.12 -0.03 -0.06 0.00

0.33 0.11 0.34 0.09 0.00 0.00

-0.14 -0.02 0.20 0.07 0.53 0.15 0.12 0.04

-0.13 -0.03 0.52 0.16 0.06 0.02 0.02 0.01

-0.15 -0.03 -0.07 0.00

"Results are shown for Cardiff (C), London (L) and Bonn (B) sets of pedigrees as well as for total (T) dataset.

The Liang test of linkage under heterogeneity is significant at P < 0.04 for both narrow and broad diagnostic models. The ESPA analysis shows significantly increased sharing of alleles ibd at PLAB amongst affected sibs in completely known sibships (i.e., where marker information, including parents, was complete) at P < 0.04, but when sibships with missing information are

included, the significance level drops to P < 0.08. The APM analysis shows increased sharing of alleles ibs at PLAB with the most significant P value of P < 0.05 using the F(p) = l/p weighting function. Thus, all three methods of analysis converge to provide evidence suggestive of linkage between a susceptibility locus for bipolar disorder and the PLA2 locus.

TABLE V. Results of Heterogeneity Tests on the Set of 45 Families for Each of the Seven Markers for N and B Diagnostic Categories

A test (homog) Liang testb

Marker Diagnostic model Max lod score Estimated cia Estimated 0" Liang statistic P value SlOl

S78

S84

N B N B N B

s79 N B

S76 N B

PLA2 N B

AFM294ze9 N B

0.00 0.00 0.67 0.69 0.15 0.38 0.23 0.43 0.02 0.26 0.60 0.91 0.00 0.00

1.00 1.00 1.00 0.75 1.0 1.0 1.00 1.00 0.50 0.85 0.90 0.8 1.00 1.00

0.5 0.5 0.2 0.1 0.3 0.2 0.3 0.2 0.3 0.2 0.2 0.1 0.5 0.5

0 0 2.93 2.48 0.64 1.88 0.81 2.12 0.01 0.30 3.52 3.34 0 0

<1.00 < L O O C0.044 <0.058 <0.22 C0.085 <0.19 <0.074 <1000 <0.29 C0.031 <0.035 <1.00 <1.00

represents proportion of families linked and 0 recombination fraction. bSee text for description of Liang test.

100 Dawson et al.

TABLE VI. Results of the ESPA Analysis for the Broad Diagnostic Model (B) for Each of the Seven Markers

No. sib Shared Non shared Marker Sibs pairs alleles alleles Chi P value SlOl CK"

PK" Total

S78 CK PK

Total S84 CK

PK Total

s79 CK PK

Total S76 CK

PK Total

PLA2 CK PK

Total AF'M294ze9 CK

PK Total

15 37 52 23 34 57 29 44 73 30 44 74 24 38 62 35 41 76 27 38 65

4 29.4 33.4 22 35.1 57.1 23 38.1 61.1 25 41.0 66.0 19 31.9 50.9 30 34.3 64.3 25 33.2 58.2

10 28.5 38.5 17 24.0 41.0 21 30.0 51.0 26 24.8 50.8 17 27.0 44.0 18 31.2 49.2 19 34.4 53.4

2.57 0.01 0.37 0.64 2.10 2.65 0.09 0.95 0.90 0.02 4.00 2.54 0.11 0.40 0.49 3.00 0.15 2.01 0.82 0.02 0.20

1<0.06Ib <0.5

[<0.29Ib <0.22 <0.08 <0.053 <0.17 <0.17 <0.17 <0.46 <0.02 <0.061 <0.38 <0.29 <0.26 <0.04 <0.38 <0.078 <0.19 <0.46 <0.33

"CK, completely known sibships; PK, partly known sibships h e . marker genotypic information must be reconstructed, at least in part, using likelihood methods). bP values in brackets reflect biologically meaningless results ke . , decreased sharing of alleles above chance expectation)

The majority of evidence in support of linkage comes from the Cardiff dataset. These 22 families were collected by a single investigator (N.C.) in the West Mid- lands of England and South Wales. Pedigree 324, which was ascertained by N.C. during the same inves- tigation, originates in North Wales. North Wales bor- ders both South Wales and the West Midlands and it could be argued that the Cardiff set of pedigrees is likely to be most genetically similar to pedigree 324 and, therefore, most likely to share a particular susceptibility locus for affective disorder. Such an hypothesis could explain the decreasing evidence in favour of linkage in the datasets from individual centres as the site of collection of the pedigrees becomes more distant from North Wales. Consistent with this possibility is the observation that the Cardiff set of pedigrees included two non-Caucasian families: one Asian and one Afro-Caribbean in origin. If these pedigrees are ex- cluded from analysis, the remaining 20 pedigrees provide a maximum lod score of 2.49 in support of linkage. In addition to the possibility of genetic heterogeneity, it is possible that clinical heterogeneity could account for the different findings by the three investigating centres. I t could be argued that the Cardiff pedigrees, being collected by the same clinical investigator as pedigree 324, may be more clinically homogeneous with each other and with pedigree 324 than are the other sets of pedigrees. However, we have not, as yet, discov- ered any clinical differences between those families giving positive and those giving negative lod scores.

If a susceptibility locus for affective disorder lies within the 12q23-q24.1 region, then there are several

potential candidate genes available for further study. Clearly, phospholipase A2 (PLA2) is of interest because it is a marker at this locus which provides the maximum evidence supporting linkage. PLAB has been suggested as playing a role in the pathogenesis of affective disorder [Hibbeln et al., 19891. However, the gene on chromosome 12 codes for a secretory form which is pre- dominantly found in the pancreas [Seilhamer et al., 1986,19891 rather than the cytosolic PLAB which is in- volved in the maintenance of membrane fluidity and composition and which has been implicated in affective disorder. Drugs affecting intracellular calcium ion con- centration, such as lithium carbonate, carbamazepine and verapamil are effective in the treatment of bipolar disorder [Dubovsky et al., 19921. Two plasma membrane Ca(2+) ATPases which play a critical role in intracellular calcium homeostasis have been mapped to this region. One isoform (ATPZB1) maps to 12q21-q23 [Olson et al., 19911 and ATP2A2 maps to 12q23-q24 [Otsu et al., 19931. The location of these genes with re- spect to the markers studied is not known.

Our finding of modest evidence in favour of linkage does not approach the conventional levels of statistical significance which would be required even for a disorder in which the mode of inheritance is well established [Ott, 19911. It could be argued that conventional significance levels are unnecessarily stringent because we had some a priori evidence suggesting that a susceptibility locus for bipolar disorder may lie within the region spanned by the markers tested. However, the strength of the a priori evidence is probably not suffi- cient to reduce the lod score criterion below 3, particularly in view of the uncertainties over the mode of in-


TABLE VII. Results of the APM Analyses for the Broad Diagnostic Category for Each of the Markers

Marker

SlOl

S78

S84

s79

S76

PLA2

AFM294ze9

T statistic”

1.40 2.60 2.48

-0.74 -0.05

0.78 0.48 0.59 0.52 0.77 0.74 0.55 0.28 0.23

-0.02 -0.46

1.16 1.70

-0.63 -0.69 -0.55

P value

<0.08 <0.005 <0.007 >0.5 >0.5 <0.22 <0.32 <0.27 <0.31 <0.23 <0.23 <0.30 <0.39 <0.41 >0.5 >0.5 <0.13 <0.05 >0.5 >0.5 >0.5

“F(p) is the weighting function and T the test statistic described by Weeks and Lange [19881.

heritance of bipolar disorder [Ott, 19911. Simulation studies suggest that a lod score of greater than or equal to 0.9 would be expected to occur under the null hypothesis of no linkage with a probability of approximately 0.015. However, we have analysed 7 markers and used two different definitions of “affected which will tend to increase the probability of a false positive finding. We therefore recommend that appropriate cau- tion be exercised when interpreting our results.

In conclusion, there is modest evidence in our dataset suggesting the possibility of linkage between bipolar disorder and markers in the region of the Darier’s disease gene in a subset of families. Although the evidence in favour of linkage is modest, this may simply reflect the relatively low power of our sample to detect linkage under heterogeneity. Our finding may, of course, represent type I error. However, the study was motivated by a specific hypothesis based upon observations in an independent pedigree (324) and, thus, by Bayes’ theorem, the evidence required to maintain our interest in this region should be lower than for a region in which there was no a priori reason to suspect a susceptibility gene. While we have not been able to provide convincing evidence for linkage, our results do not allow us to exclude the possibility that a susceptibility gene for bipolar disorder resides within this region. Given that there is some a priori evidence for the existence of such a locus, we would urge other research groups to examine markers from this chromosomal region in independent sets of pedigrees. In view of the likelihood of heterogeneity, it may prove necessary to analyse combined datasets from several centres in order to determine whether or not a susceptibility locus for bipolar disorder lies within this region of chromosome 12.

ACKNOWLEDGMENTS We thank Dr. Susan Burge for her considerable der-

matological assistance and Drs. Phil Thomas and Bobby Kurian for drawing our attention to pedigree 324. The work was supported by grants from the Beth- lem and Maudsley Research Fund (E.D.), the MRC (U.K.), the Welsh Scheme for the Development of Health and Social Research and the Commonwealth Scholarship Commission in the United Kingdom (L.L.). N.C. and M.G. are Wellcome Trust Research Fellows. M.L., W.M., M.N., P.P., and U.R. are supported by the German Research Foundation.

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