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Association of the ENGRAILED 2 (EN2) gene with autism in Chinese Han population.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 147B:434 –438 (2008)
Association of the ENGRAILED 2 (EN2) Gene
With Autism in Chinese Han Population
Lifang Wang,1,2 Meixiang Jia,1,2 Weihua Yue,1,2 Fulei Tang,1,2 Mei Qu,1,2 Yan Ruan,1,2 Tianlan Lu,1,2
Handi Zhang,1,2 Hao Yan,1,2 Jing Liu,1,2 Yanqing Guo,1,2 Jishui Zhang,3 Xiaoling Yang,1,2* and Dai Zhang1,2*
1
Key laboratory for Mental Health, Ministry of Health, P.R. China
Institute of Mental Health, Peking University, Beijing, P.R. China
3
Beijing Children’s Hospital Affiliated to Capital University of Medical Sciences, Beijing, P.R. China
2
Human ENGRAILED 2 (EN2) gene is localized to
7q36, an autism susceptibility locus. En2 knockout
mice display hypoplasia of cerebellum and a
decrease in the number of Purkinje cell, which
are similar to those reported for individuals with
autism. Furthermore, deficits in social behavior
were detected in En2/ mice. Two recent studies
have demonstrated that two intronic SNPs
(rs1861972, rs1861973) in the EN2 gene are significantly associated with autism. To investigate
whether this finding could be replicated in Chinese Han population, we performed the association study between eight single nucleotide
polymorphisms (SNPs) of the EN2 gene and autism in 210 Chinese Han trios, using the familybased association test (FBAT). The present study
demonstrated that a preferential transmission of
the rs3824068 A-allele to affected offspring (A > G:
Z ¼ 2.399, P ¼ 0.0165). After the Bonferroni correction, this statistical significance of preferential
transmission did not remain. However, when
haplotypes were constructed with multiple
markers, a number of haplotypes including three
two-marker haplotypes, nine three-marker
haplotypes, one four-marker haplotype, and one
six-marker haplotype, all of which contain the
major allele A of rs3824068, displayed significantly
associated with autism. These results were still
significant after using the permutation method to
obtain empirical P values. Thus, our data provide
evidence that the EN2 gene may be implicated in
the predisposition to autism in the Chinese Han
population.
ß 2007 Wiley-Liss, Inc.
KEY WORDS: single nucleotide polymorphism
(SNP); family-based association
test (FBAT); haplotype
Lifang Wang and Meixiang Jia contributed equally to this study.
Grant sponsor: The National Natural Science Foundation of
China; Grant numbers: 30270495; Grant sponsor: National Hightech R&D Program; Grant number: 2006AA02Z195.
*Correspondence to: Prof. Xiaoling Yang and Prof. Dai Zhang,
M.D., Ph.D., Institute of Mental Health, Peking University, 51,
Hua Yuan Bei Road, Beijing 100083, China.
E-mail: yangxl@public.fhnet.cn.net; daizhang@hsc.pku.edu.cn
Received 1 February 2007; Accepted 14 August 2007
DOI 10.1002/ajmg.b.30623
ß 2007 Wiley-Liss, Inc.
Please cite this article as follows: Wang L, Jia M, Yue W,
Tang F, Qu M, Ruan Y, Lu T, Zhang H, Yan H, Liu J,
Guo Y, Zhang J, Yang X, Zhang D. 2008. Association of
the ENGRAILED 2 (EN2) Gene With Autism in Chinese
Han Population. Am J Med Genet Part B 147B:434–438.
INTRODUCTION
Autism is a severe neurodevelopmental disorder with childhood onset that is characterized by impairments in social
interaction, communication, repetitive and stereotyped behaviors, and interests [American Psychiatric Association, 1994].
Twin and family studies indicate that genetic factors play an
important role in the etiology of autism and the estimated
heritability is over 90% [Folstein and Rutter, 1977; Bailey et al.,
1995; Szatmari et al., 1998; Folstein and Rosen-Sheidley, 2001].
The cerebellum is one of the most consistent sites of
neuroanatomic abnormality in autism. The cerebellar abnormalities including general hypoplasia and loss of Purkinje
cells, are nearly universal in the published studies of human
autopsy material [Williams et al., 1980; Ritvo et al., 1986;
Courchesne et al., 1988; Bauman, 1991; Courchesne et al.,
1994a,b; Hashimoto et al., 1995; Bailey et al., 1998; Kemper
and Bauman, 1998]. Structural MRI studies have demonstrated hypoplasia of one or more regions in the cerebellar
vermis and hemispheres [Murakami et al., 1989; Hashimoto
et al., 1995; Courchesne et al., 2001]. Furthermore, functional
MRI studies have indicated the abnormal patterning of
cerebellar activation during a motor task and attention tasks
in autism patients [Allen and Courchesne, 2003; Allen et al.,
2004]. Together, these findings from histological and imaging
studies suggest that genes involved in cerebellar development
are candidate genes in autism.
ENGRAILED 2 (EN2) gene is a homeobox transcription
factor homologous to the Drosophila melanogaster engrailed
gene with an essential role in the development of the midbrain
and cerebellum. En2 knockout and transgenic mice displayed
hypoplasia of cerebellum and decreased number of Purkinje
cell that are similar to those postmortem reported for
individuals with autism [Millen et al., 1994; Kuemerle et al.,
1997; Baader et al., 1998]. In addition, when En2 gene was
ectopically expressed in Purkinje cells, during late embryonic
and postnatal cerebellar development, the cerebellum was
greatly reduced in size and Purkinje cell numbers throughout
the cerebellum was reduced by more than one-third relative to
normal animals [Baader et al., 1998]. Misexpression of mouse
En2 in primary cortical cultures elicited a reduction in
neuronal differentiation, as reflected by the number of
process-bearing neurons that express bIII-tubulin [Benayed
et al., 2005]. Furthermore, a recent study investigated the
behavior of En2 knockout mice. Deficits in social behavior were
detected in En2/ mice across maturation that included
decreased play, reduced social sniffing and allogrooming, and
EN2 Gene and Autism
less aggressive behavior. Deficits in two spatial learning
and memory tasks were also observed [Cheh et al., 2006].
Additionally, in human beings cerebellar hypoplasia could
result from a mutation or deletion in the EN2 gene [Zec et al.,
1997; Sarnat et al., 2002]. All these results supported the
possibility that EN2 gene might be involved in the pathogenesis of autism.
Human EN2 gene is localized to chromosome 7q36.3, an
autism susceptibility locus. One Finnish study reported linkage to a combined phenotype of autism spectrum disorder and
dysphasia obtained at marker D7S550, located approximately
170 kb distal of EN2 [Auranen et al., 2002]. The other two
studies also reported the linkage for D7S483, located about
5.5 Mb proximal of EN2 locus [Liu et al., 2001; Alarcon et al.,
2002]. Additionally multiple chromosomal abnormalities
interrupting chromosome 7q have been identified in cases
with autism or autistic features. To date, studies have both
supported and refuted the validity of EN2 as a susceptibility
gene for autism [Petit et al., 1995; Zhong et al., 2003]. Two
recent family-based studies have shown that two intronic SNPs
(rs1861972, rs1861973) and the A-C (rs1861972-rs1861973)
haplotype demonstrated significant association with autism,
suggesting a role for EN2 as a susceptibility gene in the etiology
of autism [Gharani et al., 2004; Benayed et al., 2005].
In this study, we attempted to investigate the association
between the EN2 gene and autism in Chinese Han Population
using the family-based association test (FBAT).
MATERIALS AND METHODS
Subjects
The sample for this study consisted of 210 Chinese Han
family trios (singleton autistic disorder patients and their
unaffected biological parents). These families were recruited at
the Institute of Mental Health, Peking University, China. Of
the 210 autistic child probands, 196 were male and 14 were
female. The mean age of the children at the time of testing was
6.5 years (range 2–17 years). Diagnoses of autism were
established by senior psychiatrists. All patients fulfilled the
DSM-IV criteria for autistic disorder. The cases were assessed
using childhood autism rating scale (CARS) [Schopler et al.,
1980] and autism behavior checklist (ABC) [Krug et al., 1980].
Children with fragile X syndrome, tuberous sclerosis, a
previously identified chromosomal abnormality, dysmorphic
features, or any other neurological condition suspected to be
associated with autism were excluded. All subjects provided
written informed consent for participation in this study. The
study was approved by the Ethics Committee of the Health
Science Center, Peking University.
Genotyping
Genomic DNA was extracted using the phenol–chloroform
method. We selected eight single nucleotide polymorphisms
435
(SNPs) from the dbSNP (http://www.ncbi.nlm.nih.gov/SNP/)
and the international HapMap project (http://www.hapmap.org/), located at the region from exon 1 to exon 2 of EN2 gene.
These SNPs included rs3735653 (nonsynonymous change),
rs3824068, rs2361688, rs3824067, rs1861972, rs1861973,
rs4717034 (intron), and rs2361689 (synonymous change) that
span the 3.9kb. All those with frequencies of minor alleles
greater than 5% were used as genetic markers in this study.
Four SNPs (rs3735653, rs1861973, rs4717034, rs2361689)
were genotyped by polymerase chain reaction-restriction
fragment length polymorphism (PCR-RFLP) analysis and
direct DNA sequencing was used to genotype the other SNPs
(rs3824068, rs2361688, rs3824067, rs1861972). The information of primers and restriction enzymes is given in Table I. The
PCR amplification was performed in a 25 ml volume containing
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM of
each dNTP, 0.3 mM of each primer, 1 U of Taq DNA polymerase,
and 40 ng of the genomic DNA. The conditions used for PCR
amplification were an initial denaturation phase at 948C for
5 min, followed by 36 cycles at 948C for 30 sec, annealing at
62.5–688C for 30 sec, and extension at 728C for 30 sec, followed
by a final extension phase at 728C for 7 min.
Each 15 ml sample of PCR products was completely digested
with 3 U of restriction enzyme overnight. Digestion products
were separated by electrophoresis in 2% agarose gels and then
stained with ethidium bromide. Gels were read blindly by two
independent raters with discrepancies resolved by re-genotyping.
For rs3824068, rs2361688, rs3824067, and rs1861972, the
primer sequences were 50 -GTGGTTGGAAACCCAGACAGA-30
for the forward primer and 50 -TTTGGACAGGGTCGCTGTAAG-30 for the reverse primer. The PCR products were
sequenced by DNA sequencing after cleaning the PCR product
using a BigDye Terminator Cycle Sequencing Ready Reaction
Kit with Ampli Taq DNA polymerase (PE Biosystem). The
inner primers were used for the cycle-sequencing reaction, and
the fragments were separated by electrophoresis on an ABI
PRISM 377-96 DNA Sequencer (Applied Biosystem, Foster
city, CA).
Mendelian inconsistencies identified by error checking were
resolved with repeat genotyping. A number of sample genotypes could not be assigned due to repeated PCR failure or
unclear genotype results. These consist of 4 genotypes for SNP
rs3735653; 10 for SNP rs3824068, rs2361688 and rs4717034;
11 for SNP rs3824067 and rs1861972; and 4 for SNP
rs1861973.
Statistical Analysis
Deviation from the Hardy–Weinberg equilibrium (HWE) for
genotype frequency distributions was analyzed using the Chisquare goodness-of-fit test. Prior to analyses, Mendelian
inconsistencies were checked using the PEDCHECK program,
version 1.1 [O’Connell and Weeks, 1998]. Haplotype inconsistencies were identified by the SIMWALK2 program, version
TABLE I. Detailed Information of the PCR-RFLP Analysis for the Four SNPs
Primer sequence (50 ! 30 )
SNP
rs3735653
rs1861973
rs4717034
rs2361689
0
0
5 -GGGCGGCTCGTGGTGTTTCTA-3
50 -ACCGTGCAGCGAGAGCGTCTT-30
50 -AGCCGATTCATACACCGCAC-30
50 -ACCACCCTTTCCCCAGACAT-30
50 -TACCGCCATCCCTGTTCCTGA-30
50 -AGGCACCGGGTAAGGATTCTG-30
50 -CCGAGTTCCAGACCAACAG-30
50 -TCCTCACCAAGCCAACAC-30
Product
(bp)
RFLP
722
Alu I
376
PfIf I
315
BstU I
693
Eco0109 I
Allele (bp)
T
(722)
C
(62/314)
T
(315)
T
(693)
PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; SNP, single nucleotide polymorphism.
C
(135/587)
T
(62/107/207)
C
(112/203)
C
(167/526)
436
Wang et al.
2.91 [Sobel and Lange, 1996]. The power of sample size for
association tests was evaluated using the Genetic Power
Calculator program (http://statgen.iop.kcl.ac.uk/gpc/) [Purcell
et al., 2003]. For the disease locus, a prevalence of 0.001, a
genotype relative risk Aa ¼ 2 and AA ¼ 2, and a D-prime ¼ 1
were used to perform the analyses. For each marker in the
analysis, the allele frequency used was that reported in
Table II. We used the FBAT program (v. 1.5.1) (www.biostat.
harvard.edu/fbat/default.html) to perform single and multilocus tests of association [Rabinowitz and Laird, 2000]. The
FBAT program uses a generalized score statistic to perform
transmission disequilibrium tests, including haplotype analyses. Moreover, the FBAT program provides pairwise linkage
disequilibrium (LD) analysis to detect an inter-marker
relationship, using D’ value. SNP pairs were considered to be
in strong LD if D’ values were >0.70. The individual haplotype
tests were conducted under ‘‘biallelic’’ mode in haplotype
FBAT, meanwhile the global haplotype tests of association
were performed under ‘‘multiallelic’’ mode in haplotype FBAT.
FBATs were performed under an additive model. Bonferroni
correction for multiple testing was carried out to reduce type I
errors. For multilocus comparison, the permutation test has
been applied.
RESULTS
Eight SNPs in EN2 gene were genotyped in 210 Chinese Han
autism trios. The genotype distributions of the eight SNPs of
parents did not deviate from HWE (data not shown). The power
of different SNPs in our study was between 0.7501 and 0.8820.
Allele frequencies and the results of FBAT for SNPs are shown
in Table II. As for rs3824068, univariate FBAT demonstrated
that variant allele showed a preferential transmission (A > G:
Z ¼ 2.399, P ¼ 0.0165). But after the Bonferroni correction, this
statistical significance of preferential transmission did not
remain. To further analyze the pattern of linkage disequilibrium (LD) in our sample, we computed pairwise LD for all
possible combination of the eight SNPs using D’ value. Seven
SNPs (rs3735653, rs3824068, rs2361688, rs3824067,
rs1861972, rs1861973, rs4717034) were found to be in strong
LD between each other (D’ values ranging from 0.85 to 1.0)
except for rs2361689 (Table III).
In addition, the specific and global-haplotype FBAT tests of
association were performed. Positive results were showed in
Table IV. When two-marker haplotype analyses were per-
formed, three haplotypes displayed significant association.
Haplotype constructed from the A allele of rs3824068 and the C
allele of rs4717034 revealed significant excess transmission
both in the specific and global haplotype FBAT (P ¼ 0.0074
and 0.0096, respectively). The haplotype A-G (rs3824068rs2361688) and haplotype constructed from the A allele of
rs3824068 and the T allele of rs3824067 demonstrated an
excess transmission (P ¼ 0.0136 and 0.0136, respectively). In
our study three-marker haplotype analyses involving A-C
(rs3824068-rs4717034) demonstrated excess transmission
from parents to affected offspring for rs3735653-rs3824068rs4717034 (T-A-C), rs3824068-rs2361688-rs4717034 (A-G-C),
rs3824068-rs3824067-rs4717034 (A-T-C), rs3824068-rs1861972rs4717034 (A-A-C), and rs3824068-rs1861973-rs4717034 (A-C-C).
Four other haloptypes consisting of rs3824068-rs2361688
(A-G) and rs3824068-rs3824067 (A-T), respectively, and
a four-marker haplotype A-G-T-C (rs3824068-rs2361688rs3824067-rs4717034) displayed statistically significant
association (Table IV). Furthermore, when haplotype was
constructed with six markers, an A-G-T-A-C-C haplotype (rs3824068-rs2361688-rs3824067-rs1861972-rs1861973rs4717034) demonstrated significant association with autism
(Z ¼ 2.633, P ¼ 0.0085), the results were still significant when
the global haplotype FBAT was performed (w2 ¼ 10.877,
P ¼ 0.0280). These results were still significantly associated
with autism, after using the permutation method to obtain
empirical P values.
DISCUSSION
In the present study, we investigated the association of EN2
gene and autism in Chinese Han population. Eight SNPs that
span 3.9 kb of EN2 were selected. The rare allele frequencies
for the eight tested SNP were similar to those of previous study
[Benayed et al., 2005] except the SNP rs1861972 and
rs1861973. When haplotypes were constructed with two, three,
and four markers, we demonstrated that excess transmission
of a number of haplotypes from parents to affected offspring
was significant in this family-based association study. Furthermore, an A-G-T-A-C-C haplotype (rs3824068-rs2361688rs3824067-rs1861972-rs1861973-rs4717034) demonstrated
significant association with autism, the results remained
significant when the global haplotype FBAT was performed
(w2 ¼ 10.877, P ¼ 0.0280).
TABLE II. Results of FBAT for Eight SNPs in 210 Chinese Han Family Trios
Marker
rs3735653
rs3824068
rs2361688
rs3824067
rs1861972
rs1861973
rs4717034
rs2361689
Allele
Afreqa
Afreqb
Families
S
E(S)
Z
P
C
T
G
A
A
G
A
T
A
G
C
T
C
T
C
T
0.354
0.646
0.342
0.658
0.068
0.932
0.177
0.823
0.932
0.068
0.933
0.067
0.826
0.174
0.188
0.812
0.381
0.619
0.381
0.619
0.074
0.926
0.182
0.818
0.927
0.073
0.926
0.074
0.811
0.189
0.204
0.796
135
135
136
136
52
52
107
107
51
51
52
52
107
107
108
108
107.000
163.000
102.000
170.000
28.000
76.000
59.000
155.000
75.000
27.000
76.000
28.000
157.000
57.000
62.000
154.000
118.000
152.000
118.000
154.000
30.000
74.000
62.000
152.000
73.000
29.000
74.000
30.000
149.500
64.500
70.000
146.000
1.649
1.649
2.399
2.399
0.516
0.516
0.539
0.539
0.525
0.525
0.516
0.516
1.321
1.321
1.393
1.393
0.0992
0.0992
0.0165
0.0165
0.6056
0.6056
0.5900
0.5900
0.5994
0.5994
0.6056
0.6056
0.1866
0.1866
0.1637
0.1637
Values shown in bold are statistically significant.
FBAT, family-based association test; Afreq, allele frequency.
a
Allele frequency of patients.
b
Allele frequency of parents; Families, number of informative families; S, test statistics for the observed number of transmitted alleles; E(S), expected value of
S under the null hypothesis (i.e., no linkage or association).
EN2 Gene and Autism
437
TABLE III. Measure of Pairwise Linkage Disequilibrium D (D’) Between Eight SNPs in EN2 gene
rs3824068
rs2361688
rs3824067
rs1861972
rs1861973
rs4717034
rs2361689
rs3735653
rs3824068
rs2361688
rs3824067
rs1861972
rs1861973
rs4717034
0.212 (0.90)
0.040 (0.85)
0.106 (0.95)
0.039 (0.85)
0.040 (0.85)
0.015 (0.90)
0.114 (0.91)
0.044 (0.96)
0.110 (0.98)
0.044 (0.96)
0.044 (0.96)
0.108 (0.93)
0.117 (0.93)
0.012 (0.91)
0.068 (0.99)
0.069 (1.00)
0.013 (0.91)
0.014 (0.24)
0.013 (1.00)
0.012 (0.91)
0.142 (0.97)
0.122 (0.85)
0.068 (0.99)
0.013 (0.96)
0.014 (0.23)
0.013 (0.91)
0.014 (0.24)
0.122 (0.81)
The result of our study is not quite consistent with the four
previous studies that have investigated EN2 as an autism
susceptibility locus. Petit et al. [1995] reported the significant
association (P < 0.01) between a Pvu II polymorphism located
50 of the EN2 promoter and infantile autism in a case-control
study on a Northern French population. Then the second study
was performed by Zhong et al. in 196 multiplex families.
Transmission disequilibrium test did not show any association
between the nonsynonymous SNP rs3735653 from exon 1
which is only 2.9 kb from the PvuII polymorphism and autistic
disorder. There was also no linkage or association between
language and stereotypic behavior quantitative traits and the
exon 1 variant [Zhong et al., 2003].
Two recent studies have demonstrated association between
two allelic variants in the human EN2 gene and autism
spectrum disorder, suggesting a role for EN2 gene as a
susceptibility locus in autism [Gharani et al., 2004]. Their
researches demonstrated that the two intronic SNPs, rs1861972
and rs1861973, are consistently inherited more frequently in
individuals with autistic spectrum disorders (ASD) than
unaffected siblings. The A-C haplotype (rs1861972-rs1861973)
had consistently demonstrated similar of more significant
association than either SNP individually. That was initially
reported in 167 pedigrees. And the significant association was
observed in two additional data sets and in the entire sample of
518 families [Benayed et al., 2005].
However, our study did not replicate these positive results.
No significant association was obtained for SNPs rs1861972
and rs1861973. The A-C haplotype of rs1861972-rs1861973 did
not demonstrated significant association (P ¼ 0.5716). Variant
allele for rs3824068 showed a preferential transmission
(A > G: Z ¼ 2.399, P ¼ 0.0165). It is consistent with the study
by Benayed et al. [2005] that minimal association is detected
for rs3824068 individually (w2 ¼ 4.372, P ¼ 0.036). But after the
Bonferroni correction, the statistical significance of preferential transmission did not remain. In contrast, our study
demonstrated the significant excess transmission of a number
of haplotypes from parents to affected offspring.
There might be several reasons for these discordant findings.
First, our study suggests that there may be more than one
functional allele contributing to disease susceptibility in the
EN2 region and may help explain the inconsistency between
studies in the associated marker alleles. Second, a large ethnic
difference in the frequencies of the polymorphisms may exist.
Benayed et al. [2005] reported that for rs1861972 the minor
allele frequency (MAF) was 0.269 and 0.317 in the AGRE II and
NIMH data sets, respectively. And for rs1861973, the MAF was
0.279 and 0.295, respectively. Whereas in our study, the MAF
was 0.073 for rs1861972; and for rs1861973 the MAF was
0.074. So the number of informative families (at least one
parent was heterozygous) for SNP rs1861972 and rs1861973
was only 51, which decreased the ability to detect association.
This may explain the reason why we did not replicate the
positive results of previous research. Third, different ethnic
distributions could contribute to different genetic associations.
Studies from different geographical locations, across ethnic
groups are difficult to generate consistent results. Fourth,
difference in statistical methods used between the studies
should be considered [Nicodemus et al., 2007]. Moreover,
autism is a complex disease and characterized by genetic
heterogeneity.
In summary, we found no association between individual
SNPs of EN2 and autism in our family-based association study
with Bonferroni tests. However, we observed a number of
haplotypes including three two-marker haplotypes, nine threemarker haplotypes, one four-marker haplotype and one sixmarker haplotype, all of which contain the major allele A of
rs3824068, revealed significantly associated with autism.
Because haplotype has more accuracy and statistical power
than individual SNPs in LD-based association studies, it is
suggested that EN2 might be involved in the etiology of autism
in Chinese Han population. Further researches for association
TABLE IV. Haplotype Analysis for the Genetic Association Between EN2 and Autism
Specific Haplotype FBAT
Marker
Allele
rs3824068-rs4717034
A-C
rs3735653-rs3824068-rs4717034
T-A-C
rs3824068-rs2361688-rs4717034
A-G-C
rs3824068-rs3824067-rs4717034
A-T-C
rs3824068-rs1861972-rs4717034
A-A-C
rs3824068-rs1861973-rs4717034
A-C-C
rs3824068-rs2361688
A-G
rs3824068-rs2361688-rs1861972
A-G-A
rs3824068-rs2361688-rs4717034
A-G-C
rs3824068-rs3824067
A-T
rs3735653-rs3824068-rs3824067
T-A-T
rs3824068-rs3824067-rs4717034
A-T-C
rs3824068-rs2361688-rs3824067-rs4717034
A-G-T-C
rs3824068-rs2361688-rs3824067-rs1861972-rs1861973-rs4717034 A-G-T-A-C-C
Global Haplotypea FBAT
S
E(S)
Z
P
w2(df)
P
181.500
168.364
183.500
181.956
182.441
183.500
185.000
182.703
183.500
186.000
173.860
181.956
184.956
182.897
164.000
154.381
166.000
163.995
165.456
166.500
169.000
167.814
166.000
170.000
160.359
163.995
166.995
165.950
2.677
2.240
2.704
2.740
2.634
2.635
2.469
2.313
2.704
2.469
2.170
2.740
2.773
2.633
0.0074
0.0251
0.0068
0.0061
0.0084
0.0084
0.0136
0.0207
0.0068
0.0136
0.0300
0.0061
0.0056
0.0085
11.440 (3)
12.150 (5)
11.553 (4)
10.825 (3)
9.494 (4)
11.210 (4)
6.114 (2)
8.903 (3)
11.553 (4)
8.251 (3)
10.178 (4)
10.825 (3)
11.656 (4)
10.877 (4)
0.0096
0.0328
0.0210
0.0127
0.0499
0.0243
0.0470
0.0306
0.0210
0.0411
0.0375
0.0127
0.0201
0.0280
S, test statistics for the observed number of transmitted alleles; E(S), expected value of S under the null hypothesis (i.e., no linkage or association).
a
Global haplotype represents the haplotype using all possible variants.
438
Wang et al.
analysis in other samples and for the function of EN2 are
needed.
Gharani N, Benayed R, Mancuso V, Brzustowicz LM, Millonig JH. 2004.
Association of the homeobox transcription factor, ENGRAILED 2, 3,with
autism spectrum disorder. Mol Psychiatry 9:474–484.
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