AVSD, there is a hole called an ostium primum atrial
ventricular septum, and related valve defects. In
milder forms, known as partial AVSDs, there is an
ostium primum atrial septal defect often occurring
with a cleft in the anterior leaflet of the mitral valve.
Only patients with the most severe form, a complete
AVSD, were included in this study.
? 2006 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 9999:1–5 (2006)
CRELD1 Mutations Contribute to the Occurrence
of Cardiac Atrioventricular Septal Defects in
Cheryl L. Maslen,1,2,3* Darcie Babcock,1Susan W. Robinson,1Lora J. H. Bean,4
Kenneth J. Dooley,5Virginia L. Willour,6and Stephanie Sherman4
1Department of Medicine, Division of Endocrinology, Oregon Health & Science University, Portland, Oregon
2Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon
3Heart Research Center, Oregon Health & Science University, Portland, Oregon
4Department of Human Genetics, Emory University, Atlanta, Georgia
5Department of Pediatrics, Sibley Heart Center, Cardiology, Children’s Healthcare of Atlanta,
Emory University, Atlanta, Georgia
6Department of Psychiatry and Behavioral Sciences, Johns Hopkins University, Baltimore, Maryland
Received 9 June 2006; Accepted 20 August 2006
How to cite this article: Maslen CL, Babcock D, Robinson SW, Bean LJH, Dooley KJ, Willour VL, Sherman
S. 2006. CRELD1 mutations contribute to the occurrence of cardiac atrioventricular septal defects in down
syndrome. Am J Med Genet Part A 9999:1–5.
To the Editor:
Down syndrome (DS), the most common human
autosomal aneuploidy, results from trisomy of
chromosome 21. Congenital heart defects occur in
being defects in septation [Pradat, 1992; Freeman
et al., 1998; Stoll et al., 1998]. Although the spectrum
of heart defects observed in DS is varied, the
frequency of atrioventricular septal defect (AVSD)
the DS population the frequency is 2,000 in 10,000
live births, or approximately half of all congenital
heart defects [Ferencz et al., 1997].
Atrioventricular septal defect (AVSD), also known
as an atrioventricular canal defect or endocardial
cushion defect, is a congenital cardiac anomaly that
occurs when the superior and inferior endocardial
cushions fail to close completely, resulting in
incomplete formation of the atrial and ventricular
Genetic complexity and heterogeneity is a hall-
mark of AVSD. Familial cases of isolated AVSD with
clear monogenic, autosomal dominant transmission
since only 5–10% of isolated AVSD have an affected
first degree family member [Emanuel et al., 1983;
occurrence of isolated AVSDs suggests that AVSD is
trait caused by a high mutation rate in one of a few
AVSD genes [Maslen, 2004].
Two specific genetic loci for AVSD have been
identified. The AVSD1 locus on chromosome 1p31-
p21 (OMIM 606215) was delineated through char-
acterization of a family with autosomal dominant
AVSD with incomplete penetrance [Sheffield et al.,
1997]. However, the AVSD1 gene itself remains
unknown. The AVSD2 locus on chromosome 3p25
(OMIM 606217) was defined by breakpoints in the
Grant sponsor: PHS; Grant number: 5 M01 RR00334; Grant sponsor:
NIH; Grant numbers: P01 HD24605, R01 HD38979, F32 HD046337; Grant
sponsor: Children’s Healthcare of Atlanta Cardiac Research Committee;
Grant sponsor: CRC US DHS NIH; Grant number: MO1 RR00039.
*Correspondence to: Cheryl L. Maslen, Oregon Health & Science
University, L-465, 3181 SW Sam Jackson Park Rd., Portland, OR 97239.
Published online 00 Month 2006 in Wiley InterScience
tions act in a dominant manner with incomplete
penetrance and that CRELD1 is an AVSD suscept-
ibility gene that may act in concert with additional
modifier genes at other loci.
A separate study by Sarkozy et al.  found no
pathogenic mutations in an additional 31 individuals
with isolated AVSD. Collectively, patient studies
indicate that CRELD1 mutations occur in about 3%
partial AVSD have a CRELD1 mutation.
Given that CRELD1 mutations appear likely to be
genetic risk factors for AVSD in the euploid popula-
tion, we hypothesized that mutations in this gene
may contribute to the occurrence of AVSD on the
genetic background of trisomy 21. To test this
hypothesis we resequenced CRELD1 in 39 indivi-
duals with DS and a complete AVSD, which is the
two of these individuals were ascertained through a
larger study of DS aimed at understanding the cause
and resulting phenotype of trisomy 21 [Freeman
individuals were ascertained from the Sibley Heart
Center, Cardiology, Children’s Healthcare of Atlanta
specifically because they had DS and a complete
at less than 1 year of age. The remaining subjects
ranged in age from 1 to 15 years of age (mean¼
5.06 years, median¼4 years). Blood samples were
obtained from the person with DS and their parents.
Questionnaires were completed by the mother and
father to obtain demographic information about the
paternal health histories, and environmental expo-
sures. Medical records on the affected individual
were abstracted using a structured form to obtain
information about the heart defect as well as other
were required to definitively diagnose complete
AVSD. For this preliminary study, we included only
self-reported non-Hispanic Caucasians. All partici-
pants were enrolled under a protocol that was
approved by human subjects committees at all
recruitment sites. Genomic DNA was provided as
the CRELD1 gene by resequencing, as previously
described [Robinson et al., 2003]. Briefly, overlapp-
boundary junctions and at least 100 bp of intron
covering potential splicing elements, such as the
branch point sites, were generated as templates for
DNA sequence analysis. The templates were
sequenced in both directions by the OHSU General
(p.R329C) was identified showed that the mutation
was inherited from the father. The sister of the
echocardiography and color flow Doppler studies
indicated that the father and sister had structurally
and functionally normal hearts, demonstrating
incomplete penetrance of this CRELD1 variant. In a
later study Zatyka et al.  identified another
missense mutation in an individual with a partial
rare cytogenetic disorder known as 3p-syndrome
numerous syndromes that include AVSD as a
phenotypic component [for a comprehensive list
see Lin et al., 2006]. Of note, trisomy 21 is by far the
most common finding associated with AVSD.
Given the incidence of congenital heart defects in
DS there has been a great deal of focus on
chromosome 21 as a source for congenital heart
defect susceptibility genes, including those involved
in AVSD. Many studies have been based on the
premise that increased expression of chromosome
21 genes causes the features of DS, including
congenital heart defects [Deutsch et al., 2005; Mao
dosage sensitive chromosome 21 genes that con-
tribute to heart defects have focused on analysis of
individuals with segmental trisomy or monosomy 21
to identify ‘‘critical regions,’’ the smallest regions of
chromosome 21 overlap between individuals who
share a DS-associated phenotype. Resolution using
this approach is limited due to the rarity of the
condition, the complex karyotype of such indivi-
duals, which frequently includes more anomalies
than segmental trisomy, and to the heterogeneity of
the defined phenotype. For example, the ‘‘heart
defect critical region’’ which extends from 21q22.13
to 21qter between markers D21S55 and COL6A2 is
based on seven patients with a variety of heart
defects [Korenberg et al., 1992]. A narrowed heart
critical region was proposed by Barlow et al. 
that excluded several genes distal to D21S55 includ-
ing the collagen genes, COL6A1, COL6A2, and
COL18A, which other studies suggest are important
1995]. To date no single gene or set of genes on
chromosome 21hasbeenshown tocontributetothe
risk of heart defects in DS or euploid individuals.
We previously identified CRELD1 as a candidate
gene for the AVSD2 locus on chromosome 3p25
based on physical mapping to that locus and studies
demonstrating expression in the developing AV
endocardial cushions [Rupp et al., 2002]. CRELD1
encodes a cell surface protein that likely functions
as a cell adhesion molecule. A subsequent study of
50 individuals with AVSD identified three missense
mutations in CRELD1 that are specifically associated
with AVSD [Robinson et al., 2003]. Analysis of DNA
from family members of one of the probands with a
partial AVSD in which a missense mutation
Together these results suggest that CRELD1 muta-
thatalso was incompletelypenetrant.
MASLEN ET AL.
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
We previously determined that the p.R329C
alteration was not present in 400 race-relevant
control chromosomes [Robinson et al., 2003], which
has the power to detect a ?1% polymorphism with
95% confidence [Collins and Schwartz, 2002]. In
the current study we also examined DNA from
30 individuals (60 chromosomes) with trisomy 21
and absenceof congenital
documented by echocardiography. None of these
‘‘controls’’ carried the p.R329C mutation. In fact, we
donotexpect thefrequencyof CRELD1mutations to
no known relationship between CRELD1 and non-
disjunction events resulting in trisomy 21 that would
alter the allele distribution in that population.
Calcium binding EGF domains are highly con-
served with specific disulfide bonding patterns.
Consequently, addition of a free cysteine residue,
as occurs with this mutation, would be expected to
interfere with protein folding. As expected, the
p.R329C mutation alters the protein structure as
was previously shown in an analysis of recombi-
nantly expressed mutant CRELD1 [Robinson et al.,
2003]. Furthermore, this amino acid position is
conserved as an arginine residue among mammals
(Fig. 1A). Taken together, these data suggest a
specific association of the p.R329C mutation with
AVSD. The severity of the heart defect was greater in
the individual with DS, who had a complete AVSD
and additional cardiac anomalies, compared to the
affected euploid individual
partial AVSD, raising the possibility that trisomy 21
exacerbates the effect of the CRELD1 C329 allele.
The second mutation was identified in an infant
with DS and a complete balanced AVSD and
tricuspid regurgitation. There were no additional
Clinical Research Center DNA Sequencing Facility.
The sequencing electropherograms were analyzed
(by C.L.M, D.B, and S.W.R) using MutationSur-
veyorTMDNA Analysis software. All base changes
detected were heterozygous. All alterations were
confirmed by allele-specific PCR analysis as pre-
1998; Robinson et al., 2003]. The National Center for
was queried to identify DNA sequence alterations
that were commonly occurring SNPs. Population
studies were performed for alleles that appeared to
be unique (described below). Apparently unique
alleles were confirmed in a second aliquot of DNA
from the study subject, provided as an independent
sample. DNA from the parents of the subjects was
also analyzed for the presence of the mutant allele.
We identified two heterozygous missense muta-
tions in two unrelated subjects with DS and AVSD.
One was an infant that carried a recurrent mutation
a sporadic, isolated partial AVSD (ostium primum
ductus arteriosus, and tricuspid regurgitation. Of
note, there was anomalous hepatic drainage to the
right atrium. This patient also had pulmonary
hypertension. No other anomalies except for those
common in DS were noted.
of cysteine for arginine at amino acid 329 (p.R329C)
in the second calcium binding-EGF domain of the
protein [Rupp et al., 2002]. The recurrent nature of
the mutation is likely due to occurrence at a CpG
dinucleotide, creating a mutation hotspot. The
mutation was inherited from the mother, a normal
incomplete penetrance for p.R329C.
with an isolated
FIG. 1. A: Alignment of the sequence for the cb-EGF domain encoded by exon 9 from the human, bovine, dog, rat, and mouse CRELD1 genes. B: Alignment of the
identical between these species. The amino acid residues changed by the missense mutations in humans are boxed.
CRELD1 AND DOWN SYNDROME
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
Our working model to account for the greatly
increased incidence of congenital heart disease in
DS is that trisomy 21 takes the place of multiple
modifiers, so that fewer additional predisposing
mutations are required to reach a heart defect in a
person with trisomy 21. Consequently, the DS
population provides a valuable resource for the
identification and characterization of additional
susceptibility genes that contribute to congenital
heart defects, one of the most frequent congenital
anomalies among all live births.
FIG. 2. A diagrammatic representation of CRELD1 indicating the position
and identity of all known AVSD-associated mutations (white, WE domain;
horizontal hatching, EGF domains; diagonal hatching, calcium binding EGF
domains; black, two pass transmembrane domain; gray, carboxyl-terminal
domain). The mutations that were found in individuals with DSþAVSD are
underlined. The R107H mutation was found in an individual with an
unbalanced AVSD and heterotaxy, the T311I substitution is from an individual
with isolated sporadic partial AVSD, and the p.R329C mutation was originally
reported in an individual with an isolated sporadic partial AVSD [Robinson
et al., 2003] and has now been detected in an individual with DSþAVSD. The
P162A substitution was recently reported for an individual with a sporadically
occurring, isolated complete AVSD [Zatyka et al., 2005].
The alteration was a G to A transition in exon 10,
c.1240G>A, resulting in the non-conservative
substitution of a lysine for a glutamic acid residue
specific PCR analysis to assay 400 race-relevant
control chromosomes and found that the p.E414K
mutation was not present in these controls. It was
DS and no heart defect.Both parents of this proband
clinical evidence of a heart defect, although no
echocardiograms were conducted. The mutation
was not detected in either parent, indicating that it
occurred de novo in the proband, or was a germline
mutation in one of the parents. The substitution of a
mammals (Fig. 1B). Indeed, the only substitution at
that position in more distantly related species is a
conservative change to an aspartic acid residue,
indicating that an acidic residue is optimal in that
associated CRELD1 mutations identified to date.
Identification of CRELD1 mutations in 2/39 indivi-
duals (5.1%) with DS and complete AVSD suggests
that defects in CRELD1 may contribute to the
pathogenesis of AVSD in the context of trisomy 21.
This is consistent with our previous study that
demonstrated that CRELD1 mutations were asso-
ciated with AVSD and heterotaxy as well as non-
syndromic AVSD. Consequently we conclude that
CRELD1 mutations in general increase risk for
developing an AVSD, with other factors such as
ing the balance in that direction. These mutations
may be inherited or occur de novo.
The question remains as to what combination(s)
of factors is required to exceed the threshold
causing a complete AVSD. Individuals with DS have
a 2,000-fold increased occurence of complete AVSD
[Ferencz et al., 1989], making this the most highly
sensitized population to congenital heart defects.
Supported in part by PHS grant 5 M01 RR00334
HD38979 (L.J.H.B, S.L.S), F32 HD046337 (L.J.H.B),
Children’s Healthcare of Atlanta Cardiac Research
Committee (L.J.H.B, K.J.D), and CRC US DHS NIH
MO1 RR00039 (L.J.H.B, S.L.S). The authors thank Dr.
Kim Kerstann and Dr. Rob Pyatt for their contribu-
tions to this project, and Dr. Roger Reeves for his
helpful comments and guidance.
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CRELD1 AND DOWN SYNDROME
American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a
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Softproofing for advanced Adobe Acrobat Users - NOTES tool Download full-text
NOTE: ACROBAT READER FROM THE INTERNET DOES NOT CONTAIN THE NOTES TOOL USED IN THIS PROCEDURE.
Acrobat annotation tools can be very useful for indicating changes to the PDF proof of your article.
By using Acrobat annotation tools, a full digital pathway can be maintained for your page proofs.
The NOTES annotation tool can be used with either Adobe Acrobat 4.0, 5.0 or 6.0. Other
annotation tools are also available in Acrobat 4.0, but this instruction sheet will concentrate
on how to use the NOTES tool. Acrobat Reader, the free Internet download software from Adobe,
DOES NOT contain the NOTES tool. In order to softproof using the NOTES tool you must have
the full software suite Adobe Acrobat 4.0, 5.0 or 6.0 installed on your computer.
Steps for Softproofing using Adobe Acrobat NOTES tool:
1. Open the PDF page proof of your article using either Adobe Acrobat 4.0, 5.0 or 6.0. Proof
your article on-screen or print a copy for markup of changes.
2. Go to File/Preferences/Annotations (in Acrobat 4.0) or Document/Add a Comment (in Acrobat
6.0 and enter your name into the “default user” or “author” field. Also, set the font size at 9 or 10
3. When you have decided on the corrections to your article, select the NOTES tool from the
Acrobat toolbox and click in the margin next to the text to be changed.
4. Enter your corrections into the NOTES text box window. Be sure to clearly indicate where the
correction is to be placed and what text it will effect. If necessary to avoid confusion, you can
use your TEXT SELECTION tool to copy the text to be corrected and paste it into the NOTES
text box window. At this point, you can type the corrections directly into the NOTES text
box window. DO NOT correct the text by typing directly on the PDF page.
5. Go through your entire article using the NOTES tool as described in Step 4.
6. When you have completed the corrections to your article, go to File/Export/Annotations (in
Acrobat 4.0) or Document/Add a Comment (in Acrobat 6.0).
7. When closing your article PDF be sure NOT to save changes to original file.
8. To make changes to a NOTES file you have exported, simply re-open the original PDF
proof file, go to File/Import/Notes and import the NOTES file you saved. Make changes and re-
export NOTES file keeping the same file name.
9. When complete, attach your NOTES file to a reply e-mail message. Be sure to include your
name, the date, and the title of the journal your article will be printed in.