Whole genome linkage scan of recurrent depressive disorder from the depression network study.
ABSTRACT Genome-wide linkage analysis was carried out in a sample of 497 sib pairs concordant for recurrent major depressive disorder (MDD). There was suggestive evidence for linkage on chromosome 1p36 where the LOD score for female-female pairs exceeded 3 (but reduced to 2.73 when corrected for multiple testing). The region includes a gene, MTHFR, that in previous studies has been associated with depressive symptoms. Two other regions, on chromosomes 12q23.3-q24.11 and 13q31.1-q31.3, showed evidence for linkage with a nominal P < 0.01. The 12q peak overlaps with a region previously implicated by linkage studies of unipolar and bipolar disorders and contains a gene, DAO, that has been associated with both bipolar disorder and schizophrenia. The 13q peak lies within a region previously linked strongly to panic disorder. A fourth modest peak with an LOD of greater than 1 on chromosome 15q lies within a region that showed genome-wide significant evidence of a recurrent depression locus in a previous sib-pair study. Both the 12q and the 15q findings remained significant at genome-wide level when the data from the present study and the previous reports were combined.
- SourceAvailable from: Blazej Misiak[Show abstract] [Hide abstract]
ABSTRACT: Although homocysteine (Hcy) has been widely implicated in the etiology of various physical health impairments, especially cardiovascular diseases, overwhelming evidence indicates that Hcy is also involved in the pathophysiology of schizophrenia and affective disorders. There are several mechanisms linking Hcy to biological underpinnings of psychiatric disorders. It has been found that Hcy interacts with NMDA receptors, initiates oxidative stress, induces apoptosis, triggers mitochondrial dysfunction and leads to vascular damage. Elevated Hcy levels might also contribute to cognitive impairment that is widely observed among patients with affective disorders and schizophrenia. Supplementation of vitamins B and folic acid has been proved to be effective in lowering Hcy levels. There are also studies showing that this supplementation strategy might be beneficial for schizophrenia patients with respect to alleviating negative symptoms. However, there are no studies addressing the influence of add-on therapies with folate and vitamins B on cognitive performance of patients with schizophrenia and affective disorders. In this article, we provide an overview of Hcy metabolism in psychiatric disorders focusing on cognitive correlates and indicating future directions and perspectives.Frontiers in Behavioral Neuroscience 09/2014; · 4.16 Impact Factor
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ABSTRACT: Accumulating evidence suggests that inflammation plays a role in the pathophysiology of major depression. The adenosine triphosphate (ATP)-sensitive P2X7 receptor (P2X7R) plays a crucial role in microglial activation caused by inflammation. The dye brilliant blue G (BBG) is a P2X7R antagonist. This study examined whether BBG shows antidepressant effects in an inflammation-induced model of depression.Clinical Psychopharmacology and Neuroscience 04/2014; 12(1):31-6.
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ABSTRACT: To assess the association of P2RX7 gene rs2230912 polymorphism with mood disorders using a meta-analysis. DATA WERE COLLECTED FROM THE FOLLOWING ELECTRONIC DATABASES: PubMed, Excerpta Medica Database, Elsevier Science Direct, Cochrane Library, and Chinese Biomedical Literature Database, with the last report up to April 1, 2013. Odds ratio (OR) with 95% confidence interval (CI) was used to assess the strength of the association. Dependent on the results of heterogeneity test among individual studies, the fixed effect model (Mantel-Haenszel) or random effect model (DerSimonian-Laird) was selected to summarize the pooled OR. We identified 13 separate studies using search (6,962 cases and 9,262 controls). We detected significant between-study heterogeneity. No significant association of this polymorphism with mood disorders was found (P>0.05). We also performed disease-specific meta-analysis in unipolar depression and bipolar disorder. No significant association of this polymorphism with unipolar depression or bipolar disorder was found (P>0.05). Additionally, we performed subgroup analysis by different types of cases. No significant association of this polymorphism with mood disorders in clinical cohorts or population-based cohorts (P>0.05). A significant association of this polymorphism with mood disorders was found for the allele contrast in family-based cohorts (OR = 1.26, 95%CI = 1.05-1.50, P = 0.01). Overall, our meta-analysis suggests that P2RX7 gene rs2230912 polymorphism may not contribute to the risk of developing mood disorders using a case-control design. Given the discordance in the subgroup analysis by different types of cases, further studies based on larger sample size are still needed.PLoS ONE 02/2014; 9(2):e88575. · 3.53 Impact Factor
Whole genome linkage scan of recurrent
depressive disorder from the depression
Peter McGuffin1,*, Jo Knight1, Gerome Breen1, Shyama Brewster2, Peter R. Boyd2,
Nick Craddock3,11, Mike Gill4, Ania Korszun5, Wolfgang Maier6, Lefkos Middleton2, Ole Mors7,
Michael J. Owen3, Julia Perry2, Martin Preisig8, Theodore Reich9, John Rice9,
Marcella Rietschel10, Lisa Jones11, Pak Sham1and Anne E. Farmer1
1Medical Research Council Social Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King’s
College London, London SE5 8AF, UK,2GlaxoSmithKline, Research and Development, Greenford UB6 0HE, UK,
3Department of Psychological Medicine, School of Medicine, Cardiff University, Cardiff CF10 3XQ, UK,4Department
of Psychiatry, Trinity Centre for Health Sciences, Dublin 8, Ireland,5Barts and The London, Queen Mary’s School of
Medicine and Dentistry, London E1 4NS, UK,6Department of Psychiatry, University of Bonn, 53127 Bonn, Germany,
7Department of Psychiatry, University of Aarhus, DK-8000 Aarhus, Denmark,8Department of Adult Psychiatry,
University Hospital of Lausanne, 1008 Prilly-Lausanne, Switzerland,9Department of Psychiatry, Washington
University, St Louis, MO 63130, USA,10Central Institute of Mental Health, 68159 Mannheim, Germany and
11Department of Psychiatry, University of Birmingham, Birmingham, UK
Received June 2, 2005; Revised September 7, 2005; Accepted September 23, 2005
Genome-wide linkage analysis was carried out in a sample of 497 sib pairs concordant for recurrent major
depressive disorder (MDD). There was suggestive evidence for linkage on chromosome 1p36 where the
LOD score for female–female pairs exceeded 3 (but reduced to 2.73 when corrected for multiple testing).
The region includes a gene, MTHFR, that in previous studies has been associated with depressive symptoms.
Two other regions, on chromosomes 12q23.3–q24.11 and 13q31.1–q31.3, showed evidence for linkage with a
nominal P < 0.01. The 12q peak overlaps with a region previously implicated by linkage studies of unipolar
and bipolar disorders and contains a gene, DAO, that has been associated with both bipolar disorder and
schizophrenia. The 13q peak lies within a region previously linked strongly to panic disorder. A fourth
modest peak with an LOD of greater than 1 on chromosome 15q lies within a region that showed genome-
wide significant evidence of a recurrent depression locus in a previous sib-pair study. Both the 12q and
the 15q findings remained significant at genome-wide level when the data from the present study and the
previous reports were combined.
Genetic risk factors are well established for major affective
disorders, and a recent twin study has suggested that unipolar
depression has a stronger genetic influence than previously
thought. McGuffin et al. (1) have estimated that the heritability
may be .70% in a clinically ascertained twin sample, whereas
a population-based twin study resulted in a very similar esti-
mate using a re-test method of assessing lifetime diagnosis
(2). The majority of studies suggest a relative risk to siblings
(ls) of affective disorder in the region of 3 (3). However, a
recent study comparing the siblings of unipolar depressives
with the siblings of healthy controls using strict definitions
of both depression and health found a substantially higher
ls of over 9 (4). Further, the inheritance of unipolar depress-
ion is complex and involves an interplay of genetic and
# The Author 2005. Published by Oxford University Press. All rights reserved.
For Permissions, please email: firstname.lastname@example.org
*To whom correspondence should be addressed at: Medical Research Council Social Genetic and Developmental Psychiatry Centre, Institute of
Psychiatry, PO Box 80, De Crespigny Park, London SE5 8AF, UK. Tel: þ44 2078480871; Fax: þ44 2078480866; Email: email@example.com
Human Molecular Genetics, 2005, Vol. 14, No. 22
Advance Access published on October 3, 2005
environmental factors, including certain types of severe and
life-threatening events such as events associated with
humiliation or loss (5,6).
Despite the excess of females to males of approximately
two to one for unipolar depression, the heritability in a clini-
cally ascertained sample was the same in men and women
(1). Some population-based twin studies suggest at least
some of the genes conferring liability differ between the
sexes (7), whereas other studies do not (8). Although it has
been suggested that early-onset depression is more clearly
familial than later onset, this is not supported by a meta-
analysis (9). The only characteristics of probands associated
consistently with higher familiality or heritability are recur-
rence of episodes and severity of disorder (1,9).
Previous linkage studies of unipolar depression
Most previous linkage studies have been carried out in
families identified by a bipolar proband and where unipolar
and bipolar relatives are frequently grouped together into a
broad definition of affective disorders. Most such studies
have focussed on multiple affected extended pedigrees on
the assumption that there may be a subset segregating a
gene of major effect. This approach has been successful in
complex disorders such as early-onset Alzheimer’s disease
and breast cancer. However, consistent evidence of major
gene effects in bipolar disorder has not been forthcoming
(10). In addition, the unknown mode of inheritance creates
inherent difficulties in classic linkage approaches, and conse-
quently, sib-pair methods are attractive in the study of
complex familial disorders. An affected sib-pair genome
scan study of recurrent depression has now been published,
suggesting that there is a depression susceptibility locus on
chromosome 15q (11). Another genome scan focussing on
multiple affected families found the strongest evidence for
linkage on chromosome 12q (12). In addition, a genome
scan of multiple affected families with alcoholism, and in
whom, some individuals had depression or co-morbid alcohol-
ism and depression, found evidence of a depression-linked
locus on chromosome 1p (13). These results merit further
scrutiny and attempts at replication.
Allele frequencies across sites
Nominally significant allele frequency differences between
centres were detected across both depression linked and
unlinked regions, but none of these remained after Bonferroni
correction. However, as a check against biased results from
allele frequency mis-specification, we also investigated
whether incorrectly specified marker allele frequencies could
have resulted in false positive linkage signals using an
approach based on Morton’s (14) ‘pre-divided samples’ hetero-
geneity test. Parametric linkage analysis of the most signifi-
cant markers was carried out with the same allele frequencies
across centres and with centre-specific allele frequencies. The
difference in the sum of the maximum LOD score multiplied
by 4.6 is approximately a x2where the degrees of freedom
equal the number of different centres. The results provided
no evidence of heterogeneity, suggesting that observed
differences in the allele frequencies had not artificially inflated
the LOD scores.
The MOD score parametric analyses and the non-parametric
analyses using MERLIN yielded closely similar results identi-
fying the same regions, with little difference in signal size. For
the ease of presentation, only the results from MERLIN are
given here. The findings from MERLIN multipoint analyses,
where LOD scores exceeded 1, are summarized in Table 1.
Although the only LOD score greater than 3 is on chromo-
some 1p for female sib pairs, it is worth noting that there
are also positive findings on chromosomes 12q, 13q and
15q, where there is overlap with the regions previously
reported to be linked to depression or associated traits.
Chromosome 1. A peak with a maximum LOD score of ?2
overall and just over 3 in females, but with no evidence for
linkage in males, was observed on chromosome 1p36
between 13.8 and 21.8 cM and extending from 7.2 to
14.5 Mb on NCBI Build 35 (Fig. 1).
Chromosome 12. A narrow peak with a maximum LOD score
of ?1.6 overall, ?1.85 in females, was observed between
D12S1636 and D12S1583 at chromosome 12q23.3/24.11,
104.2–108.4 Mb (Fig. 2).
Chromosome 13. A peak with a maximum LOD score of ?1.5
was observed on chromosome 13q31.1–q31.3 between 75 and
87 cM and extending from 76.1 to 92.6 Mb (Fig. 3).
Chromosome 15. We observed a modest peak (two markers)
with a maximum LOD of 1.14 on chromosome 15q25.2
from 88 to 91.7 cM and from 78.7 to 84 Mb for depression
in the overall sample, but little other evidence. Although not
striking on its own, this is of interest because it lies completely
within a linkage region for depression previously reported by
Holmans et al. (11) (Fig. 4).
Parametric linkage analysis of markers on chromosomes 1,
10, 12, 13, 15, 21 and X revealed LOD scores of greater than 1
under either or both models at or near the peaks identified with
the non-parametric analysis. The result on chromosome 20
was the only non-parametric LOD of greater than 1, not con-
firmed by parametric analysis. However, this is a small signal
and may well be a false positive.
Combined analysis with previously published results
The phenotypic definitions used in the two previous whole
genome scans of depressive disorder are similar to those
used here so that it is reasonable to undertake a combined
analysis. For both chromosomes 12q and 15q, the results
from the depression network (DeNt) data set were smallest
and hence treated as the ‘secondary P-values’ (15). In the
analysis of chromosome 15, the empirical P-value was used
(rather than that derived from the test statistic). In the analysis
of chromosome 12, we have used the genome wide P-value.
This ensures appropriately conservative estimates.
3338 Human Molecular Genetics, 2005, Vol. 14, No. 22
Chromosome 12. The analysis of the DeNt data revealed a
P-value of 0.004 for marker D12S1613 at position 106.15 Mb
on NCBI Build 35, this adjusts to 0.10083. Abkevich et al.
(12) report a P-value of 0.00003 for marker D12S1706
(position 95.37 Mb). The overall P-value is therefore 0.00004.
Chromosome 15. The analysis of the DeNt data revealed a
P-value of 0.011 for marker D15S1047 at position 78.92 Mb,
this adjusts to 0.278898. Holmans et al. (11) report a P-value
of 0.023 for marker D15S652 (position 90.32 Mb). The
overall P-value is therefore 0.038803.
Our study provides support for the main positive findings from
the only whole genome scans of depression published to date
indicating loci on chromosomes 12q (12) and 15q (11). We
also have a modest linkage peak on chromosome 13q in a
region previously implicated in panic disorder and a variety
of somatic symptoms (16). This is potentially noteworthy
because there is evidence of overlap between the genes that
contribute to anxiety and depressive symptoms (17,18). It is
also worth noting that a region on chromosome 13q (15) has
been previously implicated in both bipolar disorder and
schizophrenia, and a region on 15q has been found to be
linked with lithium responsive bipolar disorder (19). How-
ever, neither of these regions overlap with those reported in
the present study.
Our most interesting new result was a peak with a
maximum LOD score of ?2 overall and just over 3 in
females, but with no contribution from male–male pairs,
observed on chromosome 1p36 between 13.8 and 21.8 cM
and extending from 7.2 to 14.5 Mb on Build 35. There have
been no previous reports of linkage on chromosome 1 in
studies of depression as such. Moreover, the region is separate
from a chromosome 1 region, reported by Nurnberger et al.
(13) to be linked with depression co-morbid with alcoholism.
Nor does it overlap with a region linked to neuroticism, a
personality trait associated with vulnerability to depression,
reported from the London center of the DeNt consortium
using a separate sib-pair sample (20). Both these are centro-
meric to the DeNt linkage region at ?60–70 cM. The findings
concerning the 1p region need to be interpreted with caution.
An LOD of 3.03 does not quite meet the guideline level for
significance of 3.3 (or 3.6 for samples consisting only of sib
pairs) suggested by Lander and Kruglyak (21) and reduces
to 2.73 if we subtract log10(2) to correct for the number of
independent tests introduced by dividing the non-parametric
analysis by sex. Strictly speaking, the result is ‘suggestive’
rather than ‘significant’.
Nevertheless, it is intriguing that a gene associated with
depression, MTHFR, encoding for the enzyme methylenetetra-
hydrofolate reductase (MTHFR) is in the middle of the DeNt
chromosome 1 linkage region (at 11.78 Mb). MTHFR cata-
lyses the conversion of 5,10-5,10-methylenetetrahydrofolate
to 5-methyltetrahydrofolate, a co-substrate for homocysteine
remethylation to methionine. Bjelland et al. (22) examined
the association between folate, total homocysteine, vitamin
B12 and an MTHFR 677C/T polymorphism and anxiety and
depression, as measured by the Hospital Anxiety and
Depression Scale, in approximately 6000 subjects. The T/T
genotype (OR, 1.69) was associated with depression but not
with anxiety and, in contrast to our finding of a linkage
signal only in women, there was no evidence of sex-specific
effects. In addition, studies by Hickie et al. (23) and
Arinami et al. (24) have found this association. There has
been one negative study (25), but it contained less than 100
depressed cases and probably lacked power to detect the
effect observed by Bjelland et al. (22). Although most of the
linkage signal comes from female–female pairs, the number
of male–male pairs is small with low power on their own to
detect an effect. Indeed, if we perform the test of Morton
(14) for linkage heterogeneity between male and female
pairs, the result is not significant (x2¼ 5.198, two degrees
of freedom, P ¼ 0.074). Furthermore, Bjelland et al. (26) in
their association study found no differences between men
and women, so that if our linkage finding largely or entirely
reflects the presence of MHTFR within this region, it is
unlikely that there are true sex differences (22).
A narrow peak with a maximum LOD of ?1.6 was
observed between D12S1636 and D12S1583 at chromosome
12q23.3/24.11, extending from 104.2 to 108.4 Mb. Thus,
it is just adjacent to the region reported as linked to
major depression by Abkevich et al. (12) on chromosome
12q22–23.2. Combined analysis of these two data sets
results in a P-value of 0.00004. It is also entirely within the
bipolar linkages reported by Craddock et al. (27) and others.
However, an important caveat is that the evidence for
linkage in the study of Abkevich et al. (12) came only from
male pairs in contrast to the present study and previously
reported bipolar study findings (26).
It is also of interest that the gene encoding D-amino acid
oxidase (DAO), that has been reported to be associated with
schizophrenia (28), lies within the region at 107.7 Mb. DAO
is expressed in the brain, where it oxidizes D-serine, a potent
Table 1. LOD scores greater than 1 from DeNt linkage scan for depression
from all pairsa
aIncluding opposite sex pairs.
Human Molecular Genetics, 2005, Vol. 14, No. 22 3339
Figure 1. Graph showing LOD scores on chromosome 1 versus centiMorgan position.
Figure 2. Graph showing LOD scores on chromosome 12 versus centiMorgan position.
3340 Human Molecular Genetics, 2005, Vol. 14, No. 22
activator of N-methyl-D-aspartate-type (NMDA) glutamate
receptors. Glutamate and aspartate are excitatory neurotrans-
mitters that have been implicated in a number of pathological
states of the nervous system. DAO has also been found to
interact with G72 protein by which it becomes activated
(27), and the G72 gene has also been implicated recently in
the bipolar affective disorder (29).
A peak with a maximum LOD score of 1.5 was observed on
chromosome 13q31.1–q31.3 between 75 and 87 cM and
extending from 76.1 to 92.6 Mb. This region is relatively
gene poor, with about half the normal density of genes and
has relatively low GC content. However, this locus is within
a region reported to be strongly linked to panic disorder. In
a study of panic disorder, Weissman et al. (30) identified a
Figure 3. Graph showing LOD scores on chromosome 13 versus centiMorgan position.
Figure 4. Graph showing LOD scores on chromosome 15 versus centiMorgan position.
Human Molecular Genetics, 2005, Vol. 14, No. 223341
group of families with a syndrome that includes frequency
of micturition, migraine and mitral valve prolapse. They
suggested that this phenotype may represent a subtype of
panic disorder. Their maximum LOD score was 3.6 at
D13S779 on chromosome 13q (?99.2 Mb) with panic as the
affected phenotype and a LOD of 4.2 was obtained when indi-
viduals with the broader syndrome were included as affected.
The same group (16) later produced a replication of this
finding with a different set of families.
Finally, we also observed a modest peak (two markers) with
a maximum LOD of 1.14 on chromosome 15q25.2 from 88 to
91.7 cM and from 78.7 to 84 Mb. Although not striking on its
own, this is of interest because it lies completely within a
linkage region for depression. Holmans et al. (11) performed
a genome scan of families multiple affected with recurrent,
early-onset major depressive disorder (MDD), thereby select-
ing a strictly defined phenotype closely similar to that used by
ourselves. The sample consisted of 297 informative families
containing 415 independent affected sib pairs. Affected cases
had recurrent MDD with onset before 31 years of age for pro-
bands or 41 years of age for other affected relatives; the mean
age of onset was 18.5 years and the mean number of depress-
ive episodes was 7.3. Genome-wide significant linkage with a
maximum LOD score of 3.7 was observed on chromosome
15q25.3–q26.2. The linkage was not sex-specific. Combined
analysis of our highest LOD score on chromosome 15 and
Holman’s highest LOD score gave a significant result at a
In conclusion, our findings provide further evidence of loci
conferring susceptibility to depression on chromosomes 12q
and 15q and lend support for the existence of a locus associ-
ated with a range of anxiety related and depressive symptoms
on chromosome 13q. Our novel linkage peak on chromosome
1p suggests that there may be depression susceptibility locus
within this region, and the region does contain a gene with a
common variant that has been implicated in depression in
three independent association studies.
MATERIALS AND METHODS
Sibling pairs affected with recurrent unipolar depression were
recruited from eight clinical sites: Aarhus, Denmark; Bonn,
Germany; Dublin, Ireland; Lausanne, Switzerland; St Louis,
USA and London, Cardiff and Birmingham, UK. In addition,
where available, parents of the affected sibling pairs were also
included in the study.
Subjects were identified from psychiatric clinics, hospitals
and general medical practices and from volunteers responding
to media advertisements. Caucasian subjects over the age of
18 were included if they had experienced two or more epi-
sodes of unipolar depression of at least moderate severity
separated by at least 2 months of remission as defined by
the Diagnostic and Statistical Manual 4th edition operational
criteria (DSM-IV) (31) or the International Classification of
Diseases 10th edition operational criteria (ICD-10), for uni-
polar depression (32). Probands were all white and of white
European parentage. They were included in the study if they
had at least one biological sibling, not a monozygotic twin,
over the age of 18 years meeting the same diagnostic criteria.
Subjects were excluded if either sibling had ever fulfilled
criteria for mania, hypomania or schizophrenia.
Subjects were also excluded if they experienced psychotic
symptoms that were mood incongruent or present when
there was no evidence of a mood disturbance. Other exclusion
criteria were intravenous drug use with a lifetime diagnosis of
dependency; depression occurring solely in relation to alcohol
or substance abuse or depression only secondary to medical
illness or medication and a clear diagnosis of bipolar disorder,
schizophrenia, schizo-affective disorder or acute or transient
psychotic disorders in first- or second-degree relatives.
The sociodemographic characteristics of subjects, their
recruitment and assessment have been described in detail by
Farmer et al. (33). Data pertinent to the present report are
summarized in Table 2.
All subjects were interviewed using the Schedules for Clinical
Assessment in Neuropsychiatry (SCAN) (34,35). Items of psy-
chopathology in the SCAN interview were rated for presence
and severity according to the worst and the second worst epi-
sodes of depression identified by the subjects. For the purposes
of rating severity, subjects were asked to identify within each
of these episodes of depression a 4–6 week period when their
symptoms were at their worst (peak intensity). The majority of
the SCAN items were coded as follows; 0—indicates the
absence of the item, 1—the item was present but to a mild
degree or intermittently throughout the peak intensity 4–6
weeks, 2—item moderately severe and present for .50% of
the peak intensity period or severe but present for ,50% of
the peak intensity period, 3—item severe for .50% of the
peak intensity period. The computerized version of the
SCAN2.1 is built on top of the IShell system, which is a
computer-aided personal interviewing tool produced by the
World Health Organization (36) and provides diagnoses
according to DSM-IV and ICD-10 operational definitions.
Interviewer training and reliability across sites
All interviewers from each site attended a 4-day SCAN
training course in the UK. Each site also undertook further
inter-rater reliability meetings regularly and annually; all
interviewers from all sites took part in a joint inter-rater
reliability exercise. Further details are provided by Farmer
et al. (33).
All sites obtained ethical approval for the DeNt study within
their own countries and institutions. All study participants
gave written informed consent for participation in the study.
At the time of the SCAN interview, interviewers obtained
25 ml of whole blood that was collected in 37.5 ml (EDTA
containing) monovettes. In addition, drops of blood were
placed on a Guthrie blood spot card. The blood samples
3342Human Molecular Genetics, 2005, Vol. 14, No. 22
were labelled with a bar code, gently mixed and stored frozen
upright in a 2208C freezer pending DNA extraction.
Phenotypic data analysis
All phenotypic information from interviews and question-
naires was coded by assigning a number to each subject and
removing any personal identifying information. The same
codes were used on the blood sample tubes, using a bar
code system. The phenotypic information was first entered
on an EXCEL spread sheet after which a data file was
created using the Statistical Package for the Social Sciences
(SPSS) version 10 for Windows for the statistical analyses.
Genotyping of microsatellite markers was carried out by
DeCode, and the results were checked for mis-specified
relationships by the programmes RELPAIR (37) and Graphi-
cal Representation of Relationships (GRR) (http:/ /www.sph.
compares the multipoint probability of the genotype data
under different possible relationships, whereas GRR calculates
the IBS mean and SD for each pair and plots these values
representing each type of relative pair using a different
colour. Decisions about each problematic pair were made on
the basis of the results from both programmes; if there was
discrepancy between the programmes, the GRR results were
used because these are less sensitive to genotyping errors.
Mendelian errors were investigated using PEDSTATS (38)
and were dealt with on a case-by-case basis. As a further
error-checking measure, MERLIN (37) was used to run analy-
sis including and excluding unlikely genotypes (i.e. those
implying double recombination in a small interval); both
options gave similar results.
If parental genotypes are missing, MERLIN calculates
identical by descent probabilities on the basis of population
allele frequencies. Therefore, it is important to check whether
there are significant differences between study sites. Allele
frequency heterogeneity between the different centres was
investigated using x2tests.
These data cleaning processes resulted in 929 individuals
from 417 families. Using an ‘N 2 1’ method of counting
affected sib pairs in sibships with more than two affecteds,
we had a total of 497 sib pairs. They consisted of 266 same-
sex female, 58 same-sex male and 173 opposite-sex pairs.
They were genotyped at 963 autosomal markers and 44 X
chromosome markers. Success rates for the autosomal
markers were .86% for 90% of the markers, with a
minimum success rate of 61%. For the X chromosome, the
success rate was .66%. For individuals, the average geno-
typing success rate was 94% with a minimum of 73% for
autosomal markers and 61% for the X chromosome.
Genotypic data analysis
Linkage. Non-parametric linkage analysis was performed
using MERLIN and MINX (http://www.sph.umich.edu/csg/
abecasis/merlin). MERLIN (37) is a multipoint engine for
rapid likelihood inference and uses sparse inheritance trees
for pedigree analysis. It performs rapid haplotyping, genotype
error detection and affected pair linkage analyses and can
handle more markers than other pedigree analysis packages.
The analysis of dichotomous trait data implemented in
MERLIN is essentially a model-free approach, where the
Kong and Cox (39) LOD score type statistic is calculated on
the basis of allele sharing. The Kong and Cox approach is
robust and highly appropriate to the DeNt sib-pair design.
However, as some DeNt sites (particularly St Louis) adopted
an ascertainment strategy that derives affected sib pairs
from extended multiplex families, there was a strong
argument for also exploring the data using an approach that
takes full advantage of larger as well as small pedigrees. In
particular, it has been proposed (40) that analysing linkage
data over a dominant model and a recessive model—in
effect maximizing the LOD score over model as well as posi-
tion—is more powerful than a model-free method and nearly
as robust. This type of the so-called MOD score analysis
was performed using the heterogeneity LOD score option
available in GENEHUNTER (41). The following parameters,
depression of ?5%, were used to specify a dominant model
q ¼ 0.05, f1¼ 0.05, f2¼ 0.5 and f2¼ 0.5 and parameters
q ¼ 0.33, f1¼ 0.05, f2¼ 0.05 and f3¼ 0.5 were used to
specify a recessive model. Parametric analysis was carried
out on the eight chromosomes that had LOD scores greater
than 1 in the non-parametric analysis. Owing to susceptibility
of multipoint parametric analysis to mis-specified allele
frequencies, single point analysis was also undertaken.
Gender-specific analyses were also performed using only
Combined data analysis. Badner and Gershon’s (15) method
was used to combine the results of chromosomes 12q and
15q with those from previous studies by Abkevich et al.
(12) and Holmans et al. (11). This method involves correcting
all but the P-value from the original study by an equation that
takes into account the distance between the peak of the initial
Table 2. Probands and affected siblings recruited from each site
SiteGender Number of
Mean age at illness
Human Molecular Genetics, 2005, Vol. 14, No. 223343
and subsequent studies (42):
adjusted P ¼ CP þ 2lGZðPÞFðZðPÞÞv½ZðPÞsqrtð4lDÞ?;
where C is the number of chromosomes, P the observed
P-value, l the rate of crossovers per Morgan, G the size of
region in Morgans, Z(P) the standard normal inverse of P
and F(Z(P)) the standard normal density function. The final
part of the equation is a correction for the fact that the test
is made at discrete points, i.e. each marker. D is the average
marker spacing in Morgans, and when Z(P)sqrt(4lD) is less
than 2, as it was in all our analysis, v can be approximated
as exp 2 (0.583x). The P-value of the original study and the
adjusted P-values of the subsequent studies are combined
using the equation given by Fisher:
where the degrees of freedom is twice the number of studies.
This study was funded by GlaxoSmithKline.
Conflict of Interest statement. M.J.O. and N.C. are consultants
to GlaxoSmithKline (GSK) and have received honoraria for
academic talks from Eli Lilly, Astra Zeneca and GSK. A.K.
has received research grants from GSK and Synthelabo-
Sanofi and has received honoraria from Eli Lilly. A.E.F. has
received honoraria for presentations and chairing meetings
from Eli Lilly, GSK and Wyeth and is a consultant for
GSK. P.M. has received honoraria from Eli Lilly and GSK
and has acted as a consultant in the recent past for GSK and
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