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Transmission disequilibrium and haplotype analyses of the G72G30 locus Suggestive linkage to schizophrenia in Palestinian Arabs living in the North of Israel.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:91 –95 (2006)
Transmission Disequilibrium and Haplotype Analyses of
the G72/G30 Locus: Suggestive Linkage to Schizophrenia in
Palestinian Arabs Living in the North of Israel
M. Korostishevsky,1 I. Kremer,2 M. Kaganovich,1 A. Cholostoy,1 I. Murad,3 M. Muhaheed,3 I. Bannoura,4
M. Rietschel,5 M. Dobrusin,6 U. Bening-Abu-Shach,1 R.H. Belmaker,6 W. Maier,5 R.P. Ebstein,7,8 and Ruth Navon1*
Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
Emek Hospital, Afula, Israel
Kemal Psychiatric Hospital, Bethlehem, Palestinian Authority
The Palestinian Research Center for Genetics of Mental Disorders, Bethlehem, Palestinian Authority
Department of Psychiatry, University of Bonn, Bonn, Germany
Beersheva Mental Health Center, Beersheva, Israel
Scheinfeld Center of Human Genetics in the Social Sciences, Department of Psychology, The Hebrew University, Jerusalem, Israel
Herzog Hospital, Jerusalem, Israel
Association of the G72/G30 locus with schizophrenia was recently reported in French Canadian,
Russian, and Ashkenazi populations using casecontrol studies. In the present study we hypothesize the existence of a G72/G30 risk allele overtransmitted to affected sibs in Palestinian Arab
families. A total of 223 Palestinian Arab families
that included an affected offspring and parents
were genotyped with 11 SNPs encompassing the
G72/G30 genes. The families were recruited from
three regions of Israel: 56 from the North (Afula),
136 from the central hill region (Bethlehem,
Palestinian Authority), and 31 from the South
(Beersheva). Individual SNP analyses disclosed a
risk allele in SNP rs3916970 by both haplotype
relative risk (HRR: x2 ¼ 5.59, P ¼ 0.018) and transmission disequilibrium test (TDT: x2 ¼ 6.03,
P ¼ 0.014) in the Afula families. Follow-up multilocus analysis using family-based association
tests (FBAT: z ¼ 2.197, P ¼ 0.028) exposed the adjacent haplotype. SNP rs3916970 is located about
8 kb from the linkage disequilibrium block that
was reported to be associated with schizophrenia
in Ashkenazi Jews. Excess of similar haplotypes of
this region was observed in the Palestinian Arabs
and the Ashkenazi patients. These data suggest a
common risk factor for schizophrenia susceptibility in the G72/G30 locus among Ashkenazi Jews
and Palestinian Arabs. The results strengthen
This article contains supplementary material, which may be
viewed at the American Journal of Medical Genetics website
Grant sponsor: Adams Super Center for Brain Studies, Tel Aviv
*Correspondence to: Ruth Navon, Department of Human
Genetics and Molecular Medicine, Sackler School of Medicine,
Tel Aviv University, Tel Aviv, Israel 69978.
Received 24 September 2004; Accepted 17 March 2005
DOI 10.1002/ajmg.b.30212
ß 2005 Wiley-Liss, Inc.
previous reports on the role of this locus in the
etiology of schizophrenia. ß 2005 Wiley-Liss, Inc.
KEY WORDS: 13q32 region; family trios; major
psychosis; multi-SNP statistics;
founder effect
Schizophrenia is a major public health problem affecting
about 1% of the population worldwide with a devastating effect
on patients’ lives. It is characterized by profound disturbances
of cognition, emotion, and social functioning. Family, twin and
adoption studies consistently show that both hereditary and
non-heritable factors influence disease susceptibility [Gottesman, 1991; Andreasen, 1995]. Being a complex disorder, it is
accepted that several genes, each with small to modest effect,
contribute to the etiology of the disease.
The identification of genes for complex disorders like
schizophrenia has been difficult as there is no definitive data
about which genes are involved. One approach to locating
candidate genes is to search for them in regions known for
linkage. A region of potential interest in schizophrenia is
chromosome 13q, suggested by a number of linkage studies
[Lin et al., 1995; Shaw et al., 1998; Levinson et al., 2000]. By
testing affected schizophrenic families, Blouin et al. [1998] and
Brzustowicz et al. [1999] narrowed the suspected region to
13q32-34 (for review see Christian et al. [2002]). However, no
candidate genes were initially observed in this region,
considered a ‘‘gene desert,’’ until Chumakov et al. [2002]
identified two overlapping genes, G72 and G30, transcribed
from opposite DNA strands. Extensive genotyping of single
nucleotide polymorphisms (SNPs) in this region, combined
with advanced statistical analysis, showed a significant
association of the G72/G30 region with schizophrenia in two
populations, French Canadians, and Russians. Subsequently,
the G72 protein was identified as a D-amino acid oxidase
activator (DAOA) [Chumakov et al., 2002]. These findings,
together with the observation that oxidation of D-serine by
DAAO greatly attenuates N-methyl-D-aspartate (NMDA)
receptor-mediated neurotransmission [Mothet et al., 2000],
are consistent with the glutamatergic theory of schizophrenia
[Konradi and Heckers, 2003]. The G30 gene is predicted to
encode a 71 amino acid protein; however, the existence of the
protein has not been verified and its function is not yet clear.
Korostishevsky et al.
TABLE I. General Characteristics of the Patient Samples
Sample size
Age of onset in
years (range)
56 (32/24)
136 (106/30)
31 (20/11)
22.0 (14–45)
24.5 (17–32)
23.0 (16–35)
Once the G72/G30 region was shown to be associated with
schizophrenia [Chumakov et al., 2002], the association had to
be replicated on different populations in independent casecontrol and family-based studies. Two independent casecontrol studies of German and Ashkenazi Jew patients
replicated the association between the G72/G30 gene locus
and schizophrenia [Korostishevsky et al., 2004; Schumacher
et al., 2004]. Intriguingly, the association to the G72/G30 gene
locus was also reported in two series of independent pedigrees
with bipolar disorder [Hattori et al., 2003].
The present study examines the involvement of the G72/
G30 gene locus with schizophrenia in Palestinian Arab
families, using transmission disequilibrium and comparative
haplotype analyses. We hypothesize the existence of a G72/G30
risk allele over-transmitted to affected sibs in the families.
Eleven SNPs in the G72/G30 region were genotyped in the 223
family trios studied.
The study enrolled 223 Palestinian Arab family trios, each
consisting of an affected offspring and parents. The families
were from three geographic regions of Israel: 56 from the North
(AF, Afula region), 136 from the center of the country (BL,
Bethlehem, Palestine authority), and 31 Bedouin families from
the South (BS, Beersheva region). All patients were diagnosed
based on the SCID interview according to DSM-IV criteria for
schizophrenia [American Psychiatric Association, 1994]. The
protocol for recruiting schizophrenic families was approved by
the local Institutional Review Board (IRB) committees. The
age of onset and the male/female distribution in the patient
samples are presented in Table I.
SNP Genotyping
Eleven SNPs were genotyped: rs3916965 (M12), rs3916966
(M13), rs3916967 (M14), rs2391191 (M15), rs3918341 (M16),
rs947267 (M18), rs778294 (M19), rs3916970 (M20), rs3916971
(M21), rs778293 (M22), and rs3918342 (M23). The designations of the SNPs in parentheses are according to Chumakov
et al. [2002]. The relative positions of the SNPs in the G72/G30
region are given on web Table V (the supplementary material).
SNP genotyping was performed by the high-throughput
system of chip-based mass spectrometry, matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) [Sequenom, Inc., San Diego, CA,]. Primer
sequences and PCR conditions are described in Korostishevsky
et al. [2004].
Re-genotyping was performed for M20, M22 in BL group and
for M22, M23 in BS group, where a deviation from Hardy–
Weinberg equilibrium (HWE) was observed. No genotyping
errors were disclosed.
Statistical Analysis
Genotype and allelic distribution at each of the 11 SNP loci
were determined by direct counting in the samples. Possible
differences in the frequency of each of the SNP genotypes or
alleles between the samples were estimated using the w2-test,
as described elsewhere [Komlos et al., 1997; Abramson and
Gahlinger, 1999]. The Arlequin software package, http://
Lgb.unige.chnarlequin [Schneider et al., 2000] was used to:
(1) evaluate the gene diversity index [Nei, 1987], (2) estimate
pairwise linkage disequilibrium (LD) between the SNP
markers [Slatkin, 1994; Slatkin and Excoffer, 1996], (3) detect
significant departure from HWE [Guo and Thomson, 1992],
and (4) calculate the maximum likelihood (ML) of haplotype
frequencies [Excoffier and Slatkin, 1995]. Based on the ML
haplotype frequency estimates, the likelihood ratio test (LRT)
[Kendall and Stuart, 1973] for sample differentiation was
applied (for details see web Appendix in the supplementary
material). Haplotype relative risk statistics (HRR) [Falk and
Rubinstein, 1987] and transmission disequilibrium test (TDT)
[Spielman et al., 1993] for individual SNPs in family trios were
performed as described by Ewens and Spielman [1995].
Haplotypic associations were tested using family-based association tests (FBAT) [Horvath et al., 2004]. The minimal
Fig. 1. Significance level for pairwise LD in the Palestinian samples. LD significance above the diagonal corresponds to AF patient sample and below the
diagonal to the total sample, AF þ BL þ BS (white: P > 0.05; gray: 0.05 < P < 0.005; dark gray: 0.005 < P < 0.0005; black: P < 0.0005).
G72 Association to Schizophrenia in Palestinians
TABLE II. ML Haplotype Frequencies in the LD Blocks of the G72/G30 Region
LD block number
Frequency (%)
SNP markers
Nucleotide sequence
Haplotypes with frequencies <5% in each sample are not enumerated; the two most frequent haplotypes in each block are in bold.
number of informative families required for a haplotype to be
included in the haplotype FBAT-test statistic was set at 10. The
Bonferroni corrections were performed by the SISA online
procedure (
Genotype, Allele, and Haplotype
Frequencies in Patient’s Samples
The patients were categorized into three subgroups according to place of dwelling. Genotype and allele frequencies for
patient samples for each SNP are summarized on web Table V
(the supplementary material). All 11 SNPs were highly
polymorphic, with an overall gene diversity of about 98% in
each of the samples (AF: 97.9 0.5, BL: 98.4 0.2, 98.4 0.6).
Pairwise LD-test disclosed three LD blocks. The LD-test
results for the AF sample and for the total sample
(AF þ BL þ BS) are presented in Figure 1.
The LD blocks were designated Ia, Ib, and II reflecting a
relatively high LD between Ia and Ib. Similar LD blocks have
been observed in other populations [Chumakov et al., 2002;
Hattori et al., 2003, Korostishevsky et al., 2004]. The haplotype
frequency estimates in the patients were performed separately
for each block (Table II). Although block Ia contains five SNPs,
only two common haplotypes, accounting for more than 90% of
all cases, occurred. Block Ib, which contains of three SNPs,
represents four haplotypes of frequent occurrence. The two
Fig. 2. Scatter-plot of HRR and TDT results. The horizontal line denotes the critical value (¼ 3.841) for 5% test significance. Scatter-plots A, B, C, and D
are for AF, BL, BS and the overall family trios, respectively.
Korostishevsky et al.
TABLE III. Results of HRR and TDT Statistics for Marker M20 in the AF Sample
most frequent haplotypes in each block are a mirror image of
each other, a phenomenon reported previously for other
populations genotyped in the G72/G30 region [Hattori et al.,
2003; Korostishevsky et al., 2004].
Family-Based Association Analyses
HRR and TDT for individual SNPs were performed in the
overall family trios as well as in AF, BL, and BS samples
separately, and the results are shown in Figure 2. Both tests
disclosed a significant deviation in allele transmission of M20
in the AF sample. An over-transmission of the A versus G
nucleotide to affected offspring was found (Table III). Other
maximal deviations in each sample, although not significant,
were observed for SNPs of block II (AF-M22, BL-M21, BSM23). The combination of all samples reduces HRR and TDT
result for M20 (Fig. 2D), while AF and BL combination
preserved near-significant values (HRR ¼ 3.69, P ¼ 0.055;
TDT ¼ 3.50, P ¼ 0.061).
Haplotype association analyses using the haplotype FBAT
statistics disclosed significant deviations for the block Ib
haplotypes in the AF sample (Table IV). Haplotype Ib-1 was
found more frequent in affected family members than expected
under the null hypothesis of no linkage and association, while
lack of haplotype Ib-4 was observed. Also multi-haplotype
FBAT permutation test disclosed significant association of the
Ib block in the AF sample (permutation cycles ¼ 31,573,
S ¼ 1,073.0, P ¼ 0.034). In BL and BS as well as in the overall
families no significant haplotype association was found (data
not shown).
The Palestinian Arab families enrolled in this study came
from three areas of the State of Israel and the Palestinian
Authority. One sample was recruited primarily from Bethlehem (presently part of the Palestinian Authority) in the
central-hill region of Israel. The second sample was from the
Negev Desert in the Southern part of Israel, the home of
the nomadic Bedouins, with Beersheva (BS) the largest city in
this region. The third group came from the Northern part of
Israel that includes the Jezreel and Galilee Valleys, with Afula
TABLE IV. Results of the Haplotype FBAT Statistics in the AF
LD block
Haplotype numbera
Haplotype numbers strictly correspond to the ones denoted in Table II.
5.59 P ¼ 0.018
6.03 P ¼ 0.014
(AF) the largest city. It was reported that Mendelian disorders
found in Palestinian Arabs are not uniformly distributed
among different geographic regions, ‘‘each of the villages may
be considered as a small isolate’’ [Zlotogora, 1997; Zlotogora,
2002]. Notably, with regard to the G72/G30 region, our
comparison of the patients from various places of dwelling
(AF, BL, and BS) revealed significant differences in the individual SNP genotype/allele frequency as well as in the haplotype frequency (web Table VI, VII respectively; in the
supplementary material).
The frequencies of individual SNP alleles transmitted and
non-transmitted to affected children were compared using
HRR and TDT (Fig. 2). TDT estimates based on minimal
nuclear families—family trios—offer an efficient way to detect
LD between a marker and a disease susceptibility gene. These
tests are similar, although the TDT, unlike HRR, focuses on
linkage rather than association, while both aspects should be
taken into account in research on a complex disorder [Ewens
and Spielman, 1995]. The SNPs of blocks Ia and II did not show
clear-cut transmission disequilibrium in the family samples,
however M20, the telomeric SNP of block Ib, exhibited
significant disequilibrium in allelic transmission (over-transmission of the A nucleotide) by both HRR and TDT in the AF
sample. The result significance is preserved after Bonferroni
correction taking into account the number of SNPs and the
inter-SNP correlation level (data not shown). The M20 SNP is
located near the 50 end of the G30 gene but outside the G72
gene. The G30 gene may regulate the G72 mRNA, a phenomenon described for other sense/antisense gene pairs [Yelin
et al., 2003].
Multilocus association tests were performed using the
haplotype FBAT statistics. This family-based statistics is
robust to population admixture and can handle unknown
haplotype phase in family trios [Horvath et al., 2004].
Haplotype Ib-1, that was found to be associated with schizophrenia in AF family sample, includes the A nucleotide at M20.
In addition, there was found an excess of haplotype II-1, in
affected family members, although not significantly. This
finding can be attributed to LD observed between M20 and the
SNPs of block II in the patient samples. Intriguingly, the very
same I-1 haplotype is the haplotype associated with schizophrenia in Ashkenazi Jews [Korostishevsky et al., 2004. To
elucidate the relation between above mentioned findings we
performed the haplotype FBAT statistics for extended M20-II
block of SNPs: M20-M21-M22-M23 (data not shown). A single
haplotype of this extended block, the A nucleotide at M20 in
addition to the II-1 alleles (A-C-A-T), demonstrates significant
association in AF families: Z ¼ 2.197, P ¼ 0.028.
This implies a common risk factor for schizophrenia
susceptibility in the G72/G30 region among Ashkenazi Jews
and Arabs. However, the involvement of this factor in
schizophrenia susceptibility differs between various ethnic
groups of Palestinian Arabs. Our findings may reflect a
common history of Jews and Arabs before the dispersion of
the Jews following the Roman conquest. Studies of classical
genomic markers, HLA, RFLPs, mitochondrial, and Y chromosome markers, as well as disease-related mutations disclosed
genetic affinities between these two populations [Hammer
et al., 2000; Nebel et al., 2000; Thomas et al., 2000].
G72 Association to Schizophrenia in Palestinians
This work was supported in part by Adams Super Center for
Brain Studies, Tel Aviv University (R.N.). Genotyping was
performed at the Genome Knowledge Center at the Weizmann
Institute of Science, supported by the Israel Ministry of Science
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