RESEARCH ARTICLE

Polymorphic Haplotypes of CRELD1 DifferentiallyPredispose Down Syndrome and Euploids Individualsto Atrioventricular Septal DefectPriyanka Ghosh,1 Pranami Bhaumik,1 Sujoy Ghosh,1,2 Umut Ozbek,3 Eleanor Feingold,3,4

Cheryl Maslen,5 Biswanath Sarkar,6 Vishmadeb Pramanik,6 Priyanka Biswas,1

Biswajit Bandyopadhyay,7 and Subrata Kumar Dey1*1Human Genetics Research Unit, School of Biotechnology and Biological Sciences, West Bengal University of Technology, Kolkata,

West Bengal, India2Department of Zoology, Sundarban Hazi Desarat College, Pathankhali, West Bengal, India3Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh. Pennsylvania4Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania5Division of Cardiovascular Medicine and OHSU Heart Research Center, Oregon Health & Science University, Portland, Oregon6DNA laboratory, Anthropological Survey of India, Kolkata, West Bengal, India7Rabindranath.Tagore International Institute of Cardiac Sciences, Kolkata, West Bengal, India

Manuscript Received: 17 February 2012; Manuscript Accepted: 26 July 2012

To explore the role of CRELD1 variants on congenital heart

defects, we sequenced the entire reading frame of CRELD1 in

the samples from Kolkata and adjoining areas. Nearly, 400 par-

ticipants were included in the genetic association study and they

were stratified as Down syndrome (DS) with atrioventricular

septal defect (AVSD), DS without AVSD, euploid with AVSD,

and euploid without AVSD. A significant association was found

between AVSD and three polymorphisms, namely rs9878047

(c.1049-129T>C), rs3774207 (c.1119C>T), and rs73118372

(c.1136T>C) among the Down syndrome and euploid individ-

uals. The polymorphism rs73118372, involves a transition

(c.1136T>C) that leads to change in amino acid methionine to

threonine which alters protein secondary structure as confirmed

by the bioinformatics software SOPMA. In addition, two haplo-

types, C-T-C andC-T-T, in the order of loci rs9878047-rs3774207-

rs73118372 were associated with incidence of AVSD among

euploid and Down syndrome, with a slightly higher odds ratio

in the later group. We hypothesize that these haplotypes increase

the risk of AVSD, and the susceptibility is exacerbated in DS,

possibly due to the trisomy 21 genetic background. Moreover, we

report for the first time on an interaction between the mutant

alleles of rs3774207 and rs73118372 which could disrupt the

delicate balance between different CRELD1 isoforms.

� 2012 Wiley Periodicals, Inc.

Key words: Down syndrome; atrioventricular septal defect;

CRELD1; single nucleotide polymorphism; splicing

INTRODUCTION

Down syndrome (DS), caused by trisomy of chromosome 21

(Ch21) is themost commonly identified genetic formof intellectual

disability characterized by multiple congenital abnormalities of

variable severity. Nearly, 50% of DS individuals suffer from a

congenital heart defect (CHD) of which approximately 40–60%have an atrioventricular septal defect (AVSD) [Freeman et al.,

1998]. Beside DS, AVSD occurs as a nonsyndromic sporadic

CHD [Digilio et al., 1999], and also part of several other syndromes

[Lin et al., 2006].

Grant sponsor: Council of Scientific and Industrial Research (CSIR), New

Delhi, India; Grant number: 27(0225)/10/EMRII.

*Correspondence to:

Subrata Kumar Dey, Human Genetics Research Unit, School of

Biotechnology and Biological Sciences. West Bengal University of

Technology, BF—142, Salt Lake City, Sector I, Kolkata, West Bengal

700064, India. E-mail: skd.humgenet@gmail.com

Article first published online in Wiley Online Library

(wileyonlinelibrary.com): 00 Month 2012

DOI 10.1002/ajmg.a.35626

How to Cite this Article:Ghosh P, Bhaumik P, Ghosh S, Ozbek U,

Feingold E, Maslen C, Sarkar B, Pramanik V,

Biswas P, Bandyopadhyay B, Dey SK. 2012.

Polymorphic haplotypes of CRELD1

differentially predisposeDown syndrome and

euploids individuals to atrioventricular septal

defect.

Am J Med Genet Part A.

� 2012 Wiley Periodicals, Inc. 1

The developmental basis of AVSD is complex. Formerly known

as an endocardial cushiondefect, basedon thepremise that it occurs

due to a failure of the atrioventricular (AV) endocardial cushions to

fuse during embryonic cardiogenesis [Eisenberg and Markwald,

1995], recent studies indicate a role for the dorsal mesenchymal

protrusion (DMP) in AV septation, and suggest that abnormalities

of the DMP could also result in AVSD. Misalignment of the atria

over the ventricles or looping abnormalities can also result in an

AVSD.

The increased prevalence of AVSD among individuals with

trisomy 21 compared to euploid individuals suggests that causative

genes for AVSD may be located on chromosome 21 including

COL6A1, COL6A2 [Davies et al., 1994, 1995; Klewer et al., 1998],

DSCAM [Barlow et al., 2001], and SLC19A1 [Locke et al., 2010].

This is further supported by a recent study showing that over-

expression of DSCAM and COL6A2 cooperatively cause atrial

defects in mice [Grossman et al., 2011]. However, regardless of

genetic background AVSD is a complex trait with genetic

heterogeneity.

CRELD1 (cysteine-rich with EGF-like domains 1; MIM 607170)

is located at the locus 3p25.3 and was originally identified as a

candidate for theAVSD2 locus [Rupp et al., 2002]. Subsequently, it

was shown that missense mutations in CRELD1 are a dominant

susceptibility factor for AVSD both in euploid [Robinson et al.,

2003; Zatyka et al., 2005] and DS [Maslen et al., 2006; Ying et al.,

2010; Kusuma et al., 2011] individuals. The gene is expressed in

embryonic cardiac progenitor cells. To gain insight into the asso-

ciation of CRELD1 polymorphisms with AVSD in both DS and

euploid individuals, we sequenced the entire reading frame of the

gene in samples obtained from subjects in Kolkata (previously

known as Calcutta) and adjoining areas.

MATERIALS AND METHODS

The studywas designed and performed following the declaration of

Helsinki and approved by the institutional ethics committee.

SubjectsFor all the case–control analyses that we performed, the participat-

ing volunteers were stratified into four categories, namely DS with

AVSD (n¼ 121), euploid with AVSD (n¼ 96), DS without any

detectable CHD(n¼ 92), and euploidwithout any detectable CHD

(n¼ 102), and for simplicity, referring to these last two classes as

‘‘groups without AVSD.’’ All subjects. mostly Bengali, were

reported randomly to our laboratory from the hospitals within

the Kolkata municipal area. The volunteers in all four categories

were chosen from hospitals of same locality to maintain the

maximum socio-demographic similarity. The cardiac phenotype

status of all subjects was ascertained by the physicians through

echocardiography; all of the case positive volunteers exhibited

complete AVSD. The average age of the DS and euploid subjects

was 7 and 9 years, respectively. The subjects were enrolled in the

study after confirmation of karyotype profile and only complete

trisomy 21 cases were included as ‘‘DS’’ for the analyses. Informed

consent was obtained for tissue sample collection and subsequent

analyses.

Genotyping of the CRELD1 GeneAll sequencing was done by individuals blinded to the case/control

and phenotype status of enrolled subjects. The sequencing was

performed in gene-walking manner, using overlapping sets of

primers covering entire length of reading frame of CRELD1. The

purified PCR amplicons were used as a template for bi-directional

DNA sequencing using the ABI PRISM 3700 DNA Analyzer plat-

form. The sequence obtained was thoroughly scanned and com-

pared against the published sequence (NCBI accession number

NM_001031717.3) of CRELD1.

Functional Prediction of Detected VariantsWe employed an in-silico approach to predict the consequences of

missense variants on protein structure and function by using the

Bioinformatics programsPolyPhen [Sunyaev et al., 2000, 2001] and

MutPred [Li et al., 2009]. Anticipation of change in secondary

structure of protein of interest was done through the SOPMA

[Combet et al., 2000] program from Network Protein Sequencing

Analysis, Pole Bioinformatics Lyonnais, France. The software

Human Splicing Finder (Version 2.4.1) was employed to predict

the effect of polymorphism on transcript splicing [Desmet et al.,

2009].

Statistical AnalysesWe tested the association between genotypes and phenotypes using

Fisher’s exact tests on2� 2or2� 3 tables, as appropriate. For all the

analyses, odds ratio was reported along with a 95% confidence

interval (CI). Difference between the SNP effects in DS and euploid

categories was tested using a logistic regression model with geno-

type, DS/euploid status and interaction as predictors; the test of

interaction terms serves as a test for the difference between DS and

Euploid Individuals.

RESULTS

Sequencing ofCRELD1 in our study cohort identified three known

SNPs, namely rs9878047, rs3774207, and rs73118372, as deter-

mined through reference sequences from NCBI and HapMap

databases. The SNP rs9878047 is located within the intron that

joins exons 8 and 9b. The second one (rs3774207) is a synonymous

variant (c.1119C>T) located in exon 9b and the third one,

rs73118372 is a nonsynonymous variant (c.1136T>C) located

in exon 9b that results in the replacement of the codon ATG

with the codon ACG, leading to change of amino acid from

methionine to threonine at the amino acid position 379 of the

CRELD1 protein (p.M379T; Table I, Fig. 1). This amino acid

substitution is predicted to be potentially deleterious to the protein

by both the MutPred and PolyPhen-2 algorithms. The SNPs

rs9878047 and rs3774207 exhibit complete linkage disequilibrium

(LD) in our results and this association is likely to be universal as we

confirmed it through HapMap published data (D’¼ 1). These

SNPs also show relatively high LD with rs73118372.

In case–control analyses for the SNP rs73118372 in the DS

group, carriers of the minor allele (non wild ‘‘C’’ allele) exhibited

more than a fourfold increased odds of AVSD (OR¼ 4.88, 95% CI

2 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

1.60–14.89, P¼ 0.0036) and in the euploid groups the odds ratio

was 3.64 (95% CI 0.95–13.92; Tables II and III). As expected given

the high LD among the SNPs, carriers of the minor allele for

rs9878047 also showed an elevated risk: OR¼ 3.44 (95% CI

1.29–9.14, P¼ 0.013) in the euploid group (Table II) and

OR¼ 4.27, (95% CI 1.87–9.74, P¼ 0.0002) in the DS group

(Table III). The degree of risk association remained identical for

the SNP locus rs3774207C>T (Tables II and III) with the geno-

types C/T and T/T being more susceptible for AVSD. We did not

observe a significant statistical difference between the odds ratios of

the DS and euploid groups (P¼ 0.46 for the interaction term in the

logistic regression model) for intronic polymorphism rs9878047.

In the second phase of analyses, we looked to see if any specific

haplotype was associated with AVSD risk, combining the above

mentioned three loci. We identified two haplotypes namely

C-T-C and C-T-T in the order of the loci rs9878047-rs3774207-

rs73118372 that are associated with risk among both the DS and

euploid samples (Table IV). The association is stronger inDS group

(P¼ 0.001) and within this category, the risk of AVSD was more

than fourfold with each copy of C-T-C haplotype compared to

T-C-T halotype (95% CI 1.60–14.89, P¼ 0.0035) and more than

three fold with each copy of C-T-T haplotype (95% CI 1.16–11.48,

P¼ 0.027; Table IV). This is highly consistent with the single-SNP

results, and suggests that the real association is likely to be with the

rs9878047-rs3774207 haplotype rather thanwith rs73118372.Hap-

lotype T-C-C (rs9878047-rs3774207-rs73118372) was not found

among both the euploid and DS samples.

Our final analysis step was in-silico functional/risk prediction.

The result of in-silico analysis as predicted byHuman Splice Finder

for the polymorphism rs3774207 (c.1119C>T) suggests a possible

deleterious effect on transcript splicing, as this synonymous change

is predicted to disrupt an exon splicing enhancer. The polymor-

phism rs7311872 (c.1136T>C) is predicted to disrupt an exon

splice silencer (ESS) and creates a new ESE (Table I, Fig. 2).

Moreover, both the programs PolyPhen and MutPred predict

an adverse effect of the non-synoymous variant c.1136T>C

(rs73118372) on CRELD1 secondary structure, which was con-

firmed subsequently in the SOPMA program.

DISCUSSION

CRELD1 is a putative cell adhesion molecule and is involved in

embryonic cardiogenesis. Previous studies have identifiedCRELD1

as a candidate gene for AVSD [Rupp et al., 2002] and mutations in

TABLE I. In Silico Inferred Effect of CRELD1 Polymorphisms Associated With AVSD

SNP locusNucleotidechange

Aminoacid change

Nature ofcodon change

Outcome of various software programs

Polyphen MutPred prediction Human splicing finderrs73118372 c.1136T> C p.M379T Non-synonymous

codingPossiblydamaging

Actionable*hypothesis

Disruption of ESS;creation of new ESE

rs3774207 c.1119C> T p.H373H Synonymous coding — — Disruption ofpotential ESE

rs9878047 c.1049-129T> C — Intronic — — —

ESE, exon splice enhancer; ESS, exon splice silencer.*Actionable hypothesis—variants with a MutPred general score (g)> 0.5 indicating the probability that the amino acid substitution is deleterious coupled with a property score P-value of P< 0.05predicting that specified structural or functional properties of the protein are impacted by the amino acid substitution.

FIG. 1. The chromatogramsshownucleotide changeat various SNP loci of CRELD1. A: Thepolymorphismat rs9878047 in the intron between the exons8

and 9b; B: Polymorphism at rs3774207 in exon 9b causes synonymous codon change; C: Polymorphism at rs73118372 of exon 9b causes

replacement of amino acid methionine to threonine.

GHOSH ET AL. 3

TABLE III. CRELD1 SNP Association With Risk of AVSD in DS Population

Polymorphic Genotype

Frequency

OR (95% CI) P valueDowns without AVSD (N¼ 92) Downs with AVSD (N¼ 121)rs9878047T/T 0.91 0.71 1.00T/C 0.07 0.24 4.186 (1.74–10.05) 0.0008C/C 0.01 0.04 4.88 (0.55–42.68) 0.21rs3774207C/C 0.91 0.71 1.00C/T 0.07 0.24 4.18 (1.74–10.05) 0.0008T/T 0.01 0.04 4.88 (0.55–42.68) 0.21rs73118372T/T 0.91 0.71 1.00T/C 0.04 0.16 4.88 (1.60–14.89) 0.0036

AVSD, atrioventricular septal defect; OR, odds ratio; CI, confidence interval.

TABLE II. CRELD1 SNP Association With Risk of AVSD in the Euploid Population

Polymorphic genotype

Frequency

OR (95% CI) P valueEuploid without AVSD (N¼ 102) Euploid with AVSD (N¼ 96)rs9878047

T/T 0.94 0.82 1.00T/C 0.04 0.12 3.64 (1.13–11.74) 0.03C/C 0.02 0.05 3.03 (0.57–16.08) 0.25

rs3774207C/C 0.94 0.82 1.00C/T 0.04 0.12 3.64 (1.13–11.74) 0.03T/T 0.02 0.05 3.03 (0.57–16.08) 0.25

rs73118372T/T 0.94 0.82 1.00T/C 0.02 0.09 3.64 (0.95–13.92) 0.07

AVSD, atrioventricular septal defect; OR, odds ratio; CI, confidence interval.

TABLE IV. Association of CRELD1 Haplotypes With AVSD Among Euploids and DS Individuals

Haplotypes

Polymorphic loci Phenotype

OR (95% CI) P valuers9878047 rs3774207 rs73118372 With AVSD Without AVSDEuploid (N¼ 198)1 T C T 0.82 0.94 1.002 C T C 0.09 0.03 3.64 (0.95–13.92) 0.073 C T T 0.08 0.03 3.24 (0.83–12.62) 0.11Down syndrome (N¼ 213)1 T C T 0.71 0.92 1.002 C T C 0.16 0.04 4.88 (1.60–14.89) 0.0033 C T T 0.11 0.04 3.566 (1.16–11.48) 0.027

OR, odds ratio; CI, confidence interval.

4 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

this gene cause AVSD in euploid [Robinson et al., 2003; Zatyka

et al., 2005] as well as in DS [Maslen et al., 2006; Ying et al., 2010;

Kusuma et al., 2011] individuals. Considering these observations

together and to gain further insights into the role of CRELD1 in

AVSDpathogenesis,we sequenced theORFs and introns of the gene

to identify any probable risk haplotypes in CRELD1.

The genetic association analyses for individual SNP loci yields a

significant association between AVSD and three polymorphisms

namely, rs9878047 (c.1049-129T>C), rs3774207 (c.1119C>T),

rs73118372 (c.1136T>C) both in euploid and DS groups (Fig. 1).

Among these, the rs73118372 (c.1136T>C) is a nonsynonymous

coding variant that causes an amino acid replacement (p.M379T;

Table I) and its deleterious effect is predicted by the Polyphen and

MutPred bioinformatics programs. The variation in the protein is

predicted to be destabilizing. The SOPMAprogram indicates that it

may result in incorporation of a secondary alpha-helical structure,

whereasMutPred predicts that it will cause a gain ofmethylation at

K378 (P¼ 0.0274) and a loss of stability (P¼ 0.0487). Therefore,

this may be the risk allele that is tagged by the other SNPs in the

haplotype block.

The haplotype study identified C-T-C and C-T-T (rs9878047-

rs3774207-rs73118372) as predisposing to AVSD both in euploid

and DS groups. There was a statistically significant association of

these two haplotypes with AVSD, with much higher penetrance

among DS (Fig. 1; Table IV). The overall risk for developing an

AVSD is 2,000 times greater in DS than euploid individuals,

indicating that trisomy 21 significantly increases susceptibility

although it is not alone sufficient to cause the defect [Maslen

et al., 2006]. The higher degree of association between susceptible

haplotypes and AVSD among DS than in euploids indicates that

there is increased penetrance of a risk allele in DS. This is consistent

with a threshold model of disease where fewer risk factors are

required to breach the disease threshold on the trisomy 21 back-

ground since it already confers substantial risk. By contrast, the

euploid genetic background presents a generally low risk for AVSD

and therefore requires a large numberof risk factors, or risk factorof

larger effect size. In other words, the effect size of this risk variant is

sufficient to push individuals with trisomy 21 past the disease

threshold, whereas it is not sufficient to do the same in lower

risk euploid individuals.

The results of the analyses involving the polymorphic site

rs3774207 are also intriguing. Although the polymorphism

(c.1119C>T) involves a synonymous codon change, it is predicted

to disrupt an exon splicing enhancer (ESE), which could result in

altered pre-mRNA splicing, excluding exon 9B from the final

transcript. The CRELD1 has multiple isoforms resulting from

alternate splicing [Rupp et al., 2002]. The SNP rs3774207 is located

in exon 9B, which is an alternately spliced ORF. The 9B isoform

eliminates the transmembranedomain and is thereforenot tethered

to the cell surface (Fig. 2). Since the protein is otherwise identical to

the cell membrane-bound version of CRELD1, it may sequester

ligand thereby modulating CRELD1 activity.

Our results suggest the existence of a potential genetic inter-

action between the SNP loci rs3774207 and rs73118372. Theminor

allele ‘‘T’’ of rs3774207disrupts anESE, so therewouldbe adecrease

in inclusion of exon 9B. On other hand, the minor allele ‘‘C’’ of

rs73118372 creates a new ESE disrupting existing exon splicing

suppressor (ESS), thereby increases the exon 9B inclusion in final

transcript (Fig. 2). However, this allele also alters the protein

configuration, causing it to be unstable. Therefore, if the minor

alleles for both of these SNPs are present together, then there is a

cumulative effect that reduces the amount of expressed CRELD1-

9B isoform. This effect could disrupt a delicate balance between 9B

and non-9B isoforms expression. These are the first potentially

pathogenic alleles in this alternatively spliced exon to be associated

with AVSD, and the first example of an association with a dis-

ruption of an ESE and ESS in CRELD1.

FIG. 2. Alternative splicing of CRELD1 nascent transcript to produce

9b and non-9b isoform proteins. The 9b isoform does not carry the

transmembrane domain and hence is not tethered to cell

membrane unlike to non-9b isoforms that carries the

transmembrane domain. In normal pre-mRNA, exon 9b harbors

both exon splicing enhancer (ESE) and exon splicing silencer

(ESS) elements rs3774207 (T> C) disrupts ESE, results in the

skipping of exon 9b left panel rs73118372 (C> T) a

nonsynonymous variant (p.M379T) disrupts the ESS and creation

of new ESE, results in inclusion of exon 9b right panel.

GHOSH ET AL. 5

In summary, we have successfully demonstrated association

between three SNPs of CRELD1 and AVSD. For the first time,

we identified two CRELD1 haplotypes associated with AVSD

phenotype among DS and euploid individuals. The apparent larger

absolute risk of the haplotype association with DS compared to

euploid individuals supports a threshold model of disease for

AVSD. Previous studies have only identified rare variants in

CRELD1 that may play a role in the etiology of AVSD. It remains

to be seen if this association also occurs in other race/ethnic groups

or is specific to the Bengali population studied here.

ACKNOWLEDGMENTS

We would like to thank the families participated in the study and

professionals who helped us in collection of tissue samples. We are

thankful to the Director, Anthropological Survey of India, Kolkata,

for providing laboratory facilities for experimental work.

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