Genetic Modifiers Predisposing to Congenital Heart …circgenetics.ahajournals.org/content/circcvg/early/2012/04/20/CIRC... · Genetic Modifiers Predisposing to Congenital Heart Disease

DOI: 10.1161/CIRCGENETICS.111.960872

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Genetic Modifiers Predisposing to Congenital Heart Disease in the

Sensitized Down Syndrome Population

Running title: Li et al.; Genetic modifiers for congenital heart disease

Huiqing Li, PhD1*; Sheila Cherry, PhD1*; Donna Klinedinst, PhD1; Valerie DeLeon, PhD2;

Jennifer Redig, PhD3; Benjamin Reshey, BS4; Michael T. Chin, MD, PhD6;

Stephanie L. Sherman, PhD7; Cheryl L. Maslen, PhD3,4,5; Roger H. Reeves, PhD1,8;

1Dept of Physiology, 2Center for Functional Anatomy, 8McKusick Nathans Inst for Genetic Med, School of Med, Johns Hopkins University, Baltimore, MD; 3Dept of Molecular & Med Genetics, 4Division of Cardiovascular Med, 5The OHSU Heart Research Center, Oregon Health & Science University, Portland, OR; 6Dept of Medicine, University of Washington School of Med, Seattle,

WA; 7Dept of Human Genetics, Emory University School of Medicine, Atlanta, GA *contributed equally

Correspondence to:

Roger H. Reeves, PhD

Department of Physiology

McKusick Nathans Institute for Genetic Medicine

School of Medicine, Johns Hopkins University

Biophysics 201, 725 N. Wolfe St.

Baltimore, MD 21205

Tel: (410) 955-6621

Fax: (443) 287-0508

E-mail: [email protected]

Journal Subject Codes: [6] Cardiac development, [130] Animal models of human disease

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Abstract:

Background - About half of people with Down syndrome (DS) exhibit some form of congenital

heart disease (CHD). However, trisomy for human chromosome 21 (Hsa21) alone is insufficient

to cause CHD as half of all people with DS have a normal heart, suggesting that genetic

modifiers interact with dosage sensitive gene(s) on Hsa21 to result in CHD. We hypothesize that

a threshold exists in both Down syndrome and euploid populations for the number of genetic

perturbations that can be tolerated before CHD results.

Methods and Results - We ascertained a group of individuals with DS and complete

atrioventricular septal defect (AVSD) and sequenced two candidate genes for CHD, CRELD1,

which is associated with AVSD in people with or without DS, and HEY2, whose mouse ortholog

produces septal defects when mutated. Several deleterious variants were identified but the

frequency of these potential modifiers was low. We crossed mice with mutant forms of these

potential modifiers to the Ts65Dn mouse model of Down syndrome. Crossing loss-of-function

alleles of either Creld1 or Hey2 onto the trisomic background caused a significant increase in the

frequency of CHD, demonstrating an interaction between the modifiers and trisomic genes. We

showed further that although either of these mutant modifiers is benign by itself, they interact to

affect heart development when inherited together.

Conclusions - Using mouse models of Down syndrome and of genes associated with congenital

heart disease we demonstrate a biological basis for an interaction that supports a threshold

hypothesis for additive effects of genetic modifiers in the sensitized trisomic population.

Key words: congenital heart disease; genetic modifier, Down syndrome

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Introduction

Congenital heart disease (CHD) is the most common congenital anomaly in humans, occurring in

as many as nine of every 1,000 infants born each year (http://www.heart.org/HEARTORG/). The

frequency is greatly elevated in people with Down syndrome (DS), as nearly half of all people

with trisomy 21 exhibit some form of CHD1. One of the more severe structural anomalies,

atrioventricular septal defect (AVSD), affects about 1/10,000 live births in the population at

large, but 1/5 individuals with DS. However, trisomy 21 alone is insufficient to cause CHD as

half of all people with DS have a normal heart, suggesting that genetic modifiers interact with

dosage sensitive gene(s) on Hsa21 to result in CHD2.

Attempts to identify dosage-sensitive Hsa21 genes that contribute to CHD have generally

focused on individuals with partial trisomy for human chromosome 21 (Hsa21) to identify

“critical regions,” the smallest regions of overlap between individuals who share a DS-associated

phenotype 3-5. However, the resolution of this approach is limited by the rarity of the condition,

the complex karyotype of such individuals (usually including other chromosomal anomalies in

addition to partial trisomy 21), and the heterogeneity of the phenotype. Several “heart critical

regions” have been defined, including a minimal region of just 1.7 Mb from DSCAM to

PKNOX1 that is based on individuals with DS and a variety of CHDs 3. To date, this kind of

analysis has not considered additional (disomic) genetic modifiers that have been associated with

CHD.

One such genetic modifier is CRELD1 (Cysteine-Rich with EGF-Like Domains 1),

initially identified as a candidate for the AVSD2 locus 6. Missense mutations in CRELD1 cause

protein misfolding 6 and are associated with AVSD in some individuals but are also present in

unaffected family members7-8. This finding suggests that CRELD1 is neither necessary nor

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sufficient to cause AVSD, but may increase the risk of developing a defect, making it a

reasonable candidate as a susceptibility locus (modifier) for AVSD. Indeed, in an earlier study

we detected mutations in CRELD1 in a small population of people with Down syndrome and

CHD2. Other genetic modifiers have been shown to affect heart development in either syndromic

individuals or model organisms. For example, somatic mutations in HEY2 have been identified

in CHD in people with Down syndrome but not in euploid populations with heart defects 9-10,

and Hey2-/- mice die in early postnatal stages from cardiac abnormalities that always include

septal defects11-12.

Animal models are crucial to our understanding of the pathogenesis of CHD and the

molecular mechanisms underlying these conditions. Orthologs of many genes on Hsa21 are

found on mouse chromosome 16 (Mmu16), with smaller subsets on Mmu10 and 1713. The most

widely used DS mouse model, Ts65Dn, is trisomic for a segment of Mmu16 containing about

half of the Hsa21 orthologs14. Ts65Dn mice display a number of the features of DS, including

cardiac abnormalities, albeit at a lower frequency than in humans15. The information derived

from mouse models suggests that, in mouse as in human, heart phenotypes likely result from a

complex genetic insult, of which dosage imbalance represents only a part. Additional genetic and

environmental factors must contribute to these phenotypes. In DS, polymorphic disomic loci on

other chromosomes may act as genetic modifiers when combined with trisomic genes.

We have created a null allele of mouse Creld1 to study the contributions of this gene to

developmental processes (Redig and Maslen, submitted). Homozygous Creld1-/- embryos die by

embryonic day (E) 11.5 and exhibit several defects, including perturbations in heart

development. In particular, the endocardial cushions form but are hypocellular, with few

mesenchymal cells evident. Little more is known about Creld1 function during development and

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how it affects heart formation. Further, there is no description of whether and how CRELD1

interacts with Hsa21 genes in normal heart development.

We hypothesize that a threshold exists, in both Down syndrome and euploid populations,

for the number of genetic perturbations that can be tolerated before CHD results (Fig. 1). In this

model, CHD in euploid individuals is caused by multiple additive factors that could be genetic,

environmental, or entirely stochastic. Given the significant increased risk for CHD in people

with trisomy 21, the DS population can be said to be "sensitized," creating a greater signal-to-

noise ratio for smaller risk factors. Identification of modifiers should therefore be enhanced in

DS using genome-wide association studies (GWAS), candidate gene sequencing in affected and

control populations, and modeling of candidate gene mutations in the mouse. Here, we provide

evidence supporting this additive hypothesis and demonstrate the utility of this approach to

confirm results from both human resequencing and mouse mutation analysis.

Materials and Methods

Patient recruiting

Human subjects were recruited nationally through the Down Syndrome Heart Project (DSHP,

http://inertia.bs.jhmi.edu/ds/index.html). Individuals with Down syndrome due to an extra intact

copy of Hsa21 verified by karyotype were included as "cases" if they had complete AV canal

(DS + AVSD) and as controls if they had DS and a normal echocardiogram. All surgical records

or echocardiograms were reviewed and confirmed by a Pediatric Cardiologist. All cases and

controls were non-Hispanic whites. Consents and procedures were approved by Institutional

Review Boards at the Johns Hopkins University School of Medicine, Emory University and

Oregon Health & Science University.

Resequencing summary

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Human CRELD1 and HEY2 genes were resequenced, including all coding regions and at least 50

bp into each flanking intron and UTRs to cover interstitial regulatory elements (Fig. 2 and

Supplemental table 1). Analyses were done by standard DNA sequencing of PCR-amplified

genomic DNA by the Oregon Clinical Translational Research Institute sequencing core facility.

Electropherograms were transferred electronically for detailed analysis facilitated by the

MutationSurveyor software suite (Soft Genetics). All sequences were compared to a reference

sequence, and all variants were noted and further analyzed. Variants of interest were confirmed

by resequencing a newly-prepared amplicon. With one exception, all variants reported were

heterozygous (see Table 1).

Variants were categorized by the type of variant and documented SNPs were annotated

by query of the SNP database. We compared SNP frequency to identify any allele-specific

disease associations. Variants were categorized as missense, insertion/deletion, non-coding,

splice site, regulatory, and other, and prioritized for disease-association potential (i.e., the

likelihood that the variant will alter gene expression or protein product structure or function).

Missense variants were analyzed using the Polyphen (http://genetics.bwh.harvard.edu/pph/) and

SIFT (http://sift.jcvi.org/) algorithms for predictions of likelihood of “damage” to the protein

product. We also monitored lack of expected SNP occurrence as an indicator of gene deletion.

All variants were analyzed in control individuals (DS without heart defects) using DNA

resequencing of the amplicon encompassing the alteration, allele-specific PCR analysis (ASP) or

restriction enzyme digestion as previously described7.

Animal Husbandry

Mice were maintained in an AAALAS-certified clean facility with food and water ad libitum.

Ts65Dn mice (B6EiC3H-a/A-Ts65Dn, Jackson Laboratory) were maintained on the B6;C3H

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background. Mice used in this study were the progeny of female Ts65Dn mice. Creld1 null mice

were generated by gene targeting (Redig and Maslen, submitted; see Supplementary Fig. 1) and

backcrossed a minimum of 8 generations onto a C57BL/6J background. Mice bearing a null

allele of Hey2 were generated by gene targeting16. All procedures were approved by the

Institutional Animal Care and Use Committee.

Genotyping

Genomic DNA was extracted from tail tips of mice and was used for genotyping by PCR.

Ts65Dn mice were identified by both PCR and fluorescence in situ hybridization (FISH) as

described17-18.

Genotypes of Creld1 and Hey2 knockout mice were determined by PCR. For Creld1

genotyping, two sets of primers that amplify the deleted region of Creld1 and a portion of the

neomycin resistance gene were added together in each reaction. The sequences of the primers for

Creld1 genotyping are as follows: E1-2F: 5’-CATCCTTCTCCCCGAGCTGAG-3’; G2-F: 5’-

CCAGTCAAAAACCACAGAGAGGG-3’; E1-2R: 5’-GTGTTTCCACCCCCGAAGT-3’.

PCR was done under the following cycling conditions: 95 C 1 min, (94 C 30s, 68 C 30s, 72 C 1

min) for 3 cycles, (94 C 30s, 67 C (-1 C/cycle) 30s, 72 C 1 min) for 10 cycles, (94 C 30s, 58 C

30s, 72 C 1 min) for 25 cycles, 72 C 5 min.

For Hey2 genotyping, two sets of primers that amplify the deleted region of Hey2 and a

portion of the neomycin resistance gene were added together in each reaction. The sequences of

the primers for Hey2 genotyping are as follows: pK065A: 5’-

CACTAAGAACTAGCGATCTGG-3’; pGK: 5’-GCACGAGACTAGTGAGACGTG-3’; CHF-

1WT3P: 5’-CTCAGGGGATTTTGAAAGC-3’. The PCR was done under the following cycling

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1 C/cycle) 30s, 68 C 1 min) for 16 cycles, (94 C 30s, 52 C 30s, 68 C 1 min) for 25 cycles, 68

C 5 min.

Histology

The progeny of various crosses were collected at P0 within hours of birth. Pups were euthanized

and thoraxes were removed and fixed in 10% formalin. Tissues were embedded in paraffin and

sectioned at 7μm by standard methods, followed by staining with Hematoxylin and Eosin using

standard methods. Heart morphology for each animal was analyzed under a dissecting

stereomicroscope by at least two individuals blinded to genotypes to evaluate the frequency and

severity of phenotypes. Photos were taken using a Nikon Digital Sight system.

Real-time analysis of gene expression

Total RNA was extracted from the L-cells or the hearts of four-week-old mice with different

genotypes by using TRIzol reagent (Invitrogen). cDNA synthesis was carried out with the First-

Strand cDNA synthesis kit (Life Sciences) using 8 μg of total RNA as template. PCR was carried

out using Taqman Gene Expression Assays (Applied Biosystems) on a 7500 Real-Time PCR

System (Applied Biosystems). For Creld1 and Hey2 co-regulation study, fluorescent- (FAM-)

labeled Creld1, Hey2, Notch1 and Gapdh were normalized to a VIC-labeled internal control, -

actin. Gapdh was used as a negative control. All comparisions refer to the WT.

Cell culture and Western blotting

L-cells were a gift of Dr. Gerry Weinmaster and were grown in DMEM with high glucose and

10% FBS, supplemental non-essential amino acids and Penicillin/Streptomycin (Invitrogen) at

37°C and 5% CO2. The pCS2+/Hey2 construct was kindly provided by Dr. Manfred Gessler.

The pCS2+ or pCS2+/Hey2 construct was transfected into the L-cells by Lipofectamine Ltx

(Invitrogen). Forty-eight hours later, half of the transfected cells were harvested and total RNA

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was extracted; the other half was lysed in RIPA buffer (Invitrogen) for Western Blot. The anti-

HEY2 antibody was purchased from PROTEINTECH (Cat. #10597-1-AP) and the anti-Actin

antibody from DSHB (JLA20).

Statistical Analysis

Mendelian inheritance of alleles and trisomies were assessed by Chi-square tests. The

prevalence of heart defects for different mouse genotypes was compared by Fisher’s exact test

using GraphPad Prism version 5. The relative quantification of gene expression from different

genotypes was compared by Mann-Whitney test. All tests were two-tailed and p-values of

p<0.05 were considered significant.

Results

Candidate gene mutations in a sensitized population.

We identified genes that could be involved in congenital heart disease based on data from either

human populations or mouse experimental data and chose two, CRELD1 and HEY2, for

resequencing in "cases" (individuals with DS and complete AVSD). The heart condition was

defined by strict criteria including two independent assessments of surgical notes for AVSD or a

normal echocardiography report. CRELD1 was resequenced in 135 cases (DS+AVSD), including

39 cases from a previous study 2 and 96 new cases. Four individuals each contained one of three

different missense variants (Table 1 and Fig. 2). One variant (p.V13M) is a known SNP

(rs279552) that has been seen in equal numbers of non-syndromic cases and controls and is

predicted to be benign with regard to protein structure/function. The previously described

p.R329C mutation was found in 2 cases, one reported before and the other is a new occurrence in

the greatly expanded population in this study. This variant was identified originally in

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individuals with non-syndromic AVSD 7 and has not been identified in over 400 control

chromosomes2, 7. The p.R329C variant has been shown to affect CRELD1 protein structure 7. In

each case the p.R329C mutation was inherited from an unaffected parent. The third missense

variant, p.E414K, predicted to be damaging to the protein, was identified in one case among the

39 indivudals examined previously and was not detected in 200 control chromosomes.

As part of our candidate gene study we also resequenced HEY2 in 90 cases (Table 1). A

single coding region variant was identified (p.L196L) which appears to be a relatively common

SNP. Four non-coding variants of unknown significance were identified. All are predicted to be

benign. For both HEY2 and CRELD1, all variants were heterozygous.

Reduced expression of Creld1 increases septal defects in Ts65Dn mice.

Human association studies7-8 and our resequencing analysis in a sensitized DS population

indicate that inactivating mutations in CRELD1 may increase the likelihood of developing a

septal defect in individuals with DS. We hypothesized that reduced expression of Creld1 would

interact with trisomic genes in a mouse DS model to increase the occurrence of heart defects.

Based on this hypothesis, we created mice carrying a null allele of Creld1 that reduces gene

expression to about 50% of normal in heterozygotes. No expression is detected in Creld1-/-

embryos (Supplemental Fig. 1). Creld1+/- mice were crossed to Ts65Dn, and progeny were

collected within hours of birth, prepared for histology and assessed for the presence of septal

defects. All classes of progeny were recovered at expected frequencies (Supplemental Table 3).

A recent study detected septal defects in 1 out of 18 similarly analyzed newborn (P0)

Ts65Dn mice15. We expanded this observation and saw a single septal defect among 25 Ts65Dn

mice at P0 (combined frequency of septal defects = 2/43 or 4.7%) (Table 2). Creld1+/- itself had

no obvious effect on phenotype and none of the Creld1+/- pups that we analyzed at P0 was found

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to have a septal defect (n=45; 18 were on the B6;C3H background of Ts65Dn mice and 27 were

inbred on B6). However, the frequency of septal defects increased dramatically to 33% (6/18) in

Ts65Dn, Creld1+/- mice (Table 2). Among those pups with heart defects, three had ostium

secundum ASDs and three had membranous VSDs (Table 2 and Fig. 3). Thus, there is a

significant difference in the frequency of heart defects between either Ts65Dn (p=0.006) or

Creld1+/- (p=0.0003) mice and those with both genetic conditions (Ts65Dn, Creld1+/-). This

observation demonstrates a biological basis for a phenotype-altering interaction between the

effects of trisomic genes and reduced Creld1 expression, and delimits a region orthologous to

part of Hsa21q22.1 - the trisomic segment in Ts65Dn - trisomy for which is sufficient to

predispose to CHD.

Reduced expression of Hey2 increases septal defects in trisomic mice.

Somatic variants of HEY2 have been identified in hearts from humans with septal defects9. In

Hey2-/- mice, the reported frequency (penetrance) of septal defects varies with genetic

background11-12, 16, 19-20, reaching more than 80% on an inbred C57Bl/6J (B6) background. We

detected membranous VSDs in eleven out of fourteen Hey2 null neonates (79%).

We crossed a null allele of Hey2 onto the Ts65Dn trisomic background. Ts65Dn, Hey2+/-

pups were underrepresented among the progeny, suggesting a deleterious additive effect of

reduced Hey2 expression with the trisomic genes from Ts65Dn (Supplemental Table 3). A single

null allele of Hey2 was not sufficient to cause septal defects (0 out of 45). However, the

frequency of septal defects was significantly increased in Ts65Dn, Hey2+/- mice, with six out of

twenty-five (24%) displaying a septal defect (p=0.04 or 0.0014 compared with Ts65Dn or

Hey2+/-, respectively) (Table 2).

Like Creld1, expression variants of Hey2 act as a modifier, contributing to CHD in a

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manner that is detectable on a sensitized trisomic background but not in euploid mice. Of note,

membranous VSDs are the only septal defect in Hey2-/- mice, but we detected additional

structural defects in Ts65Dn, Hey2+/-. Among the six pups with heart defects, 3 had membranous

VSDs, 1 had a muscular VSD, and two had ostium secundum ASDs (Table 2 and Fig. 3). Thus

the pattern as well as the number of defects is altered by the Hey2: trisomy interaction.

Combining benign Creld1 and Hey2 modifier variants results in septal defects.

To test our initial hypothesis that additive effects of modifier genes contribute to CHD on a

euploid background, Creld1+/- mice were crossed with Hey2+/-. Genotypes of progeny were

recovered at the expected frequencies (Supplemental table 3). No septal defects were detected at

P0 in 90 pups that were either Creld1+/- (n=45) or Hey2+/- (n=45), however, three out of thirty-

one double heterozygous Creld1+/-, Hey2+/- pups had a septal defect (Table 2). This result

indicates that Creld1+/- and Hey2+/- interact to increase the risk of CHD (p=0.016, Fisher's exact

test, two-tailed).

Further support for an interaction comes from the observation that while homozygosity

for a null allele in Hey2 -/- mice results exclusively in membranous VSD, all three defects in the

double heterozygotes were ostium secundum ASDs. We performed a second cross of a double

heterozygous Creld1+/-, Hey2+/- mouse to Hey2+/-. Ten pups were recovered that were

homozygous for the Hey2 null allele (which is expected to produce membranous VSD) and

heterozygous at Creld1 (Table 2). Of the ten, one had a normal heart and nine had membranous

VSDs; for those nine mice with membranous VSDs, one also had an ostium secundum ASD and

one had a muscular VSD in addition to membranous involvement. This result suggests that

reducing Creld1 expression affected the pattern of defects in homozygous Hey2 null mice,

providing further support for an interaction between Creld1 and Hey2.

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Creld1 interaction with Hey2

Creld1 encodes a cell-surface protein and Hey2 is a nuclear transcription factor. Both are

normally expressed in heart during development and throughout life. We assessed transcript

levels of both genes in hearts of mutant mice. As expected, Creld1 RNA expression was reduced

in Creld1+/- mice, but surprisingly, so was the expression of Hey2 (Fig. 4). The reverse was true,

as well; Hey2 and Creld1 both showed reduced expression in hearts of Hey2+/- mice. We saw no

difference in expression of Creld1 or Hey2 in Ts65Dn compared to euploid hearts from mice that

did not have the Creld1 or Hey2 null alleles.

To explain the co-regulation of Creld1 and Hey2, we considered the Notch signaling

pathway. Binding of a ligand to the Notch receptor causes cleavage and release of a

transcriptional activator domain that, with further processing, is transported to the nucleus to

activate transcription of target genes, including Hey2 21 . Since Hey2 expression was down-

regulated in Creld1+/- mice, we assessed the transcript level of Notch1 and found it was also

down-regulated in these mice (Fig. 4). This raises the possibility that Creld1 might affect Hey2

expression through the Notch pathway.

Since Creld1 was down-regulated in Hey2 +/- mice, we also tested whether Hey2 might be

a transcription factor for Creld1 22. Creld1 is expressed in L-cells. Transfection of a

pCS2+/Hey2 construct into L-cells significantly increased the Creld1 mRNA expression by 1.9

fold compared to cells transfected with pCS2+ alone (Fig. 5).

Discussion

Heart development is complex and a complete genetic explanation for isolated CHD, which is

generally believed to be a multifactorial condition, has proven to be a challenge. However, it is

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clear from extensive work with mutant mice that genetic background (modifiers) has a

significant influence on both the type and the frequency of heart defects for a given mutation.

For example, mice carrying mutant alleles of Tbx5, Nkx2.5 or Hey2 show different outcomes

both in terms of prevalence and also in changes in the timing or location of the maldevelopment

when the same mutant allele is bred onto different genetic backgrounds 20, 23-24. The high

incidence but incomplete penetrance of CHD in the DS population suggests that variations at

disomic loci, i.e., on chromosomes other than Hsa21, may act as genetic modifiers in

combination with trisomic genes. In other words, DS, the leading risk factor for CHD in general

and septal defects in particular, represents a sensitizing genetic condition.

Numerous studies in mouse models have shown that genetic background can affect the

severity of heart defects24. For example, the phenotype and survival rate of Hey2-/- mice is

strongly influenced by genetic background, with the highest mortality rates observed on

C57BL/6 or 129Sv/J inbred backgrounds 11-12, 16, 19-20. The correlation of variable phenotypes

with different inbred backgrounds supports the idea that (disomic) allelic variants modify Hey2

function. Similarly, heterozygous Nkx2.5 knockout mice frequently have septal defects when the

mutation is bred onto the B6 background, but the prevalence is substantially reduced in a first-

generation (F1) outcross to FVB/N or A/J24.

In people, CRELD1 mutations have been specifically associated with AVSD2, 8.

However, this may be an ascertainment bias since most studies have focused on AVSD, and

more partial AVSDs have been analyzed than complete AVSDs. Most CRELD1 missense

mutations have been found in individuals with a partial AVSD (ostium primum ASD), occurring

in approximately 2% (5/253) of cases from multiple studies7, 25-27. CRELD1 mutations are also

found in about 2.5% (4/159) of complete AVSD cases studied, including heterotaxy-associated

ondition.

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AVSD and AVSD in Down syndrome 7, 25, 28. There were no CRELD1 mutations identified in a

study of 110 individuals with isolated ostium secundum ASD 29.

Although only membranous VSD is reported in Hey2-/- mice16, 19-20, introduction of a null

allele of Hey2 into Ts65Dn mice caused muscular VSD and ASD in addition to membranous

VSD. Further, the structural defect noted in Hey2+/-, Creld1+/- mice was secundum ASD. It will

be instructive to determine whether timing or levels of expression, or the small structural

differences in Creld1 or Hey2 proteins between mouse and human might contribute to these

differences. Either way, a requirement for regulated Creld1 and Hey2 expression to achieve

normal septation is clearly demonstrated here.

It should be possible to further localize the dosage sensitive (trisomic) genetic modifiers

present on the T65Dn marker chromosome by introducing Creld1 or Hey2 mutations onto other

trisomic models of Down syndrome with smaller segmental trisomies, such as Ts65Dn/Ms1Rhr,

Ts1Cje and Ts1Rhr30-31. These mice over-express subsets of the genes upregulated in Ts65Dn

mice. The absence of AVSD in Ts65Dn mice may reflect the fact that they are trisomic for

orthologs of only about half of Hsa21 genes. Mouse models trisomic for Hsa21-orthologous

segments of Mmu17 and 10 and for all of the Hsa21 orthologs on Mmu16 will prove useful to

further characterize the contributions of dosage sensitive genes to CHD13, 32-33. These then

become candidates for resequencing in the DS case and control populations described here.

The Ts65Dn populations generated in these studies will also be useful in the genome-

wide search for disomic modifiers. As in our DS case and control human populations, we have

generated trisomic Ts65Dn, Creld1+/- mice with and without septal defects that can be used for a

genome-wide study of QTLs that explain the presence or not of CHD on this background.

The two disomic modifiers, Creld1 and Hey2, showed co-regulation at the transcript

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level. These alterations might involve the Notch signaling pathway since Notch1 is a major

transcription factor for Hey2. In that regard it is interesting that the structure of CRELD1, a

predicted transmembrane protein with four EGF-like repeats in its extracellular domain6, is

reminiscent of the non-canonical Notch ligands, DNER 34 and DLK-135. Like CRELD1, both of

these lack the DSL motif of canonical Notch ligands but contain tandem EGF repeats. We tried

to dtermine whether Creld1 is a non-canonical ligand of Notch1. However, we could not

demonstrate a physical interaction of Creld1 and Notch1 via co-IP nor a functional one using a

Notch transcription reporter assay 36 (data not shown).

Our results support a threshold hypothesis in which multiple genetic variants that are

themselves benign can interact in an additive manner to produce a structural defect in the heart

(Fig. 1). We identify two such candidates, CRELD1 and HEY2. The deleterious effects of

mutations in CRELD1 that were themselves insufficient to produce CHD were uncovered in

human beings while Hey2 effects were first demonstrated in mice. Biological proof for

interactions of both genes with trisomy and for direct interactions on a euploid background was

obtained using mouse models. In combination, the benign single variants exhibit additive effects

resulting in CHD in euploid individuals. This is one of several examples of how the genetic

legacy of people with DS contributes to the health of the general population.

Acknowledgements: We thank Eleanor Feingold for assistance with statistical analysis of gene sequence and Jonathan Epstein for expert instruction in heart phenotyping. Manfred Gessler and Gerry Weinstein graciously provided mice or reagents. We thank Darcie Babcock for technical assistance and Ken Dooley for his review of medical records to establish heart status.

Funding Sources: This work was supported in part by Award Number R01HL083300 from the National Heart, Lung, and Blood Institute (RHR, CLM, SLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung and Blood Institute or the National Institutes of Health. Additional support was from

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the Oregon Clinical and Translational Research Institute (OCTRI), grant number UL1 RR024140 from the National Center for Research Resources (CLM).

Conflict of Interest Disclosures: RHR is a scientific advisor to Roche Pharmaceuticals and to Elan Pharmaceuticals. Neither of these relationships involves the work described here.

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MMMMMMMMMMMMMMMMMMMMMMMMMM, , , , , , DaDaDaDaDDaDaDaDaDaDaDaDaDaDaDaDDaDaDaDaDaDDDaDD i iiiiii iiiiiiii L,L,L,L,LL,L,L,L,L,L,L,L,L,L,L,LLL,LL,L,L,L, eeee eee eeee e eeeet ttttt alaalalalalalalalallalaaaaaaaalolutioiiooioioooioooiioooooon n nnnnnnn n nnnnnn n n n ananananananananananananananannanannalalaalalaalalalaalalaalaalalalysysysyysysysysysysysysysysysysysyyysyyyssysy iiiiiisiiiiiiiii

P N tl A d S i U S A 2009 106 12031 12036

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24. Winston JB, Erlich JM, Green CA, Aluko A, Kaiser KA, Takematsu M, et al. Heterogeneity of genetic modifiers ensures normal cardiac development. Circulation. 2010;121:1313-1321.

25. Guo Y, Shen J, Yuan L, Li F, Wang J, Sun K. Novel creld1 gene mutations in patients with atrioventricular septal defect. World J Pediatr. 2010;6:348-352.

26. Sarkozy A, Esposito G, Conti E, Digilio MC, Marino B, Calabro R, et al. Creld1 and gata4 gene analysis in patients with nonsyndromic atrioventricular canal defects. Am J Med Genet A. 2005;139:236-238. 27. Zatyka M, Priestley M, Ladusans EJ, Fryer AE, Mason J, Latif F, et al. Analysis of creld1 as a candidate 3p25 atrioventicular septal defect locus (avsd2). Clin. Genet. 2005;67:526-528. 28. Kusuma L, Dinesh SM, Savitha MR, Krishnamurthy B, Narayanappa D, Ramachandra NB. A maiden report on creld1 single-nucleotide polymorphism association in congenital heart disease patients of mysore, south india. Genet Test Mol Biomarkers. 2011;15:483-487. 29. Posch MG, Perrot A, Schmitt K, Mittelhaus S, Esenwein EM, Stiller B, et al. Mutations in gata4, nkx2.5, creld1, and bmp4 are infrequently found in patients with congenital cardiac septal defects. Am J Med Genet A. 2008;146A:251-253. 30. Sago H, Carlson EJ, Smith DJ, Kilbridge J, Rubin EM, Mobley WC, et al. Ts1cje, a partial trisomy 16 mouse model for down syndrome, exhibits learning and behavioral abnormalities. Proc Natl Acad Sci U S A. 1998;95:6256-6261. 31. Olson LE, Richtsmeier JT, Leszl J, Reeves RH. A chromosome 21 critical region does not cause specific down syndrome phenotypes. Science. 2004;306:687-690. 32. Pereira PL, Magnol L, Sahun I, Brault V, Duchon A, Prandini P, et al. A new mouse model for the trisomy of the abcg1-u2af1 region reveals the complexity of the combinatorial genetic code of down syndrome. Hum Mol Genet. 2009;18:4756-4769. 33. Yu T, Li Z, Jia Z, Clapcote SJ, Liu C, Li S, et al. A mouse model of down syndrome trisomic for all human chromosome 21 syntenic regions. Hum Mol Genet. 2010;19:2780-2791. 34. Eiraku M, Tohgo A, Ono K, Kaneko M, Fujishima K, Hirano T, et al. Dner acts as a neuron-specific notch ligand during bergmann glial development. Nat Neurosci. 2005;8:873-880. 35. Baladron V, Ruiz-Hidalgo MJ, Nueda ML, Diaz-Guerra MJ, Garcia-Ramirez JJ, Bonvini E, et al. Dlk acts as a negative regulator of notch1 activation through interactions with specific egf-like repeats. Exp Cell Res. 2005;303:343-359. 36. Lindsell CE, Shawber CJ, Boulter J, Weinmaster G. Jagged: A mammalian ligand that activates notch1. Cell. 1995;80:909-917.

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20

Table 1. CRELD1 and HEY2 sequencing results.

Gene Variants Detected (dbSNP rs #) Amino acid change SIFT/Polyphen

predictions Minor allele

frequency (MAF) in DS cases

MAF in controls (in dbSNP)

NM_001031717.2:c.1-770G>A (rs279551)

NA (5 UTR) †60/270 0/110 (0.009)

NM_001031717.2:c.1-756C>G NA (5 UTR) 1/270 0/110

NM_001031717.2:c.1-550C>T NA (5 UTR) 1/270 0/110

NM_001031717.2:c.261+41C>T NA (intron 2) 1/270 0/110

NM_001031717.2:c.111+96C>A NA (intron 3) 2/270 0/110

NM_001031717.2:c.985C>T R329C Intolerant/Possibly Damaging

2/270 *0/400

NM_001031717.2:c.37G>A (rs279552) V13M Tolerant/Benign 1/270 (0.004) 1/132 (0.006)

NM_001031717.2:c.945G>A (rs76764016)

P315P 4/270 0/110 (0.009)

NM_001031717.2:c.1104G>A Q368Q 1/270 1/110

CRELD1

NM_001031717.2:c.1240G>A E414K Intolerant/Possibly Damaging

1/270 0/200

NM_012259.2:c.588G>C (rs61737181) L196L 20/180 (0.111) 8/100 (0.056)

NM_012259.2:c.84-94C>G (rs2875881) NA (intron 1) 63/180 (0.350) 37/100 (0.403)

NM_012259.2:c.163-13T>C NA (intron 2) 1/180 (0.006) 0/100

NM_012259.2:c.246+39C>G NA (intron 3) 6/180 (0.033) 1/100

HEY2

NM_012259.2:c.328+6T>C (rs1935978) NA (intron 4) 12/180 (0.067) 1/110 (0.054)

MAF – Minor allele frequency NA – not in coding region; coding variants shown as single letter amino acid code *200 race-matched control chromosomes without DS (Robinson et al, 2003) + 200 control chromosomes with DS (Maslen et al, 2006) †All 30 individuals are GG genotype MAF in controls; calculated from frequency in study controls; MAF from dbSNP when available

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21

Table 2. Frequency of heart defects on mutant and trisomic genetic backgrounds.

Phenotype Genetic background % of affected With septal

defectWithout septal

defect Type of septal defect

Ts65Dn B6/C3H§ 4.7% 2 41 2 membranous VSD B6/C3H 0 0 18 N/A Creld1+/-

B6 0 0 27 N/A B6/C3H 0 0 20 N/A Hey2+/- B6 0 0 25 N/A

Ts65Dn, Creld1+/- B6/C3H§ 33.3%* 6 12 3 membranous VSDs, 3 secundum ASDs

Ts65Dn, Hey2+/- B6/C3H§ 24%† 6 19 3 membranous VSDs,

1 muscular VSD, 2 secundum ASDs

Creld1+/-, Hey2+/- B6 9.7%‡ 3 28 3 secundum ASDs Hey2-/- B6 78.6% 11 3 11 membranous VSDs

Creld1+/-, Hey2-/- B6 90% 9 1

9 membranous VSDs, among those 9, 1 has extra secundum

ASD and 1 has extra muscular VSD

* indicates a significant difference between Ts65Dn, Creld1+/- and Ts65Dn (p=0.006) or Creld1+/- (p=0.0003); † significant difference between Ts65Dn, Hey2+/- and Ts65Dn (p=0.04) or Hey2+/- (p=0.0014); ‡ significant difference between Creld1+/-, Hey2+/- and the single mutants (p=0.016). §75% B6, 25% C3H; 50% B6, 50% C3H;

11111111999 99999 999 999999

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22

Figure Legends:

Figure 1. A threshold model for CHD. We hypothesize that the human population includes

allelic variants in multiple genes that contribute to the risk of congenital heart disease, many of

which have subtle or no effects by themselves. Additive effects of individual modifier genes can

reach a threshold whereby heart septal development is disrupted (Eu with CHD), but the

likelihood of inheriting many predisposing modifiers is small. Trisomy 21 is a significant risk

factor for CHD, but alone is not sufficient to produce heart defects. However, those people with

an extra copy of Hsa21 may require fewer disomic or trisomic modifiers to reach the threshold

(Ts21 + Modifier). The relative contribution of the modifier in the sensitized DS population is

therefore more readily detectable.

Figure 2. Resequencing strategy for CRELD1 and HEY2. We assessed coding regions of

these genes including all exons with 50+ bp of sequence in the flanking introns, the 5 UTR and

50+ bp of the 3 UTR. A) Resequencing strategy for CRELD1; B) Resequencing strategy for

HEY2. For exact coordinates, see Supplemental Table 1.

Figure. 3. A variety of septal defects were observed in mutant and trisomic mice at P0. A)

Normal heart showing intact ventricular septum at P0; B) membranous VSD; C) muscular VSD ;

D) normal heart showing atrial septum; E) ostium secundum ASD; F) ASD at higher

magnification. For the incidence of defects in various models, see Table 2. Arrows indicate

communication between the chambers. RA: right ventricle; LV: left ventricle; RA: right atrium;

LA: left atrium; Scale bars: A-E, 400 μm, f, 150 μm.

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23

Figure 4. Notch1, Creld1 and Hey2 interaction. A TaqMan assay was used to show coordinate

regulation of Creld1 and Hey2 transcript levels in hearts of heterozygous null mice of either

genotype. Notch1 was significantly down-regulated in Creld1+/- hearts, as well. Fluorescent-

(FAM-) labeled Creld1, Hey2, Notch1 and Gapdh were normalized to a VIC-labeled internal

control, -actin. Gapdh was used as a negative control. . The number of hearts tested was 4-6 per

analysis as indicated on the dot plot. All statistical comparisons are relative to WT. * p<0.05; **

p<0.01 (Mann-Whitney test).

Figure 5. Hey2 can activate Creld1 expression in L-cells. A) Western blot showing Hey2

protein expression in the L-cells transfected with pCS2+/Hey2 but not in control cells transfected

with pCS2+ alone . An antibody to -actin was used as a loading control. B) TaqMan assay

showed a 1.9-fold increase of Creld1 mRNA expression in pCS2+/Hey2 transfected L-cells

compared to controls. Plotted values are the average ratios of Creld1 mRNA expression in seven

experiments. Std. deviation is indicated. p value is indicated (Mann-Whitney test).

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Michael T. Chin, Stephanie L. Sherman, Cheryl L. Maslen and Roger H. ReevesHuiqing Li, Sheila Cherry, Donna Klinedinst, Valerie DeLeon, Jennifer Redig, Benjamin Reshey,

PopulationGenetic Modifiers Predisposing to Congenital Heart Disease in the Sensitized Down Syndrome

Print ISSN: 1942-325X. Online ISSN: 1942-3268 Copyright © 2012 American Heart Association, Inc. All rights reserved.

TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics

published online April 20, 2012;Circ Cardiovasc Genet.

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CIRCCVG/2011/960872 /R1

SUPPLEMENTAL MATERIAL

Supplemental Table 1.

CRELD1 was amplified by PCR from genomic DNA as 6 amplicons that span the 10 exons of

the gene, and includes intron-exon boundaries and at least 50 bp into each intron from the 5′

and 3′ boundaries and untranslated regions (UTR). Genomic coordinates are from the National

Center for Biotechnology Information Consensus Coding Sequence Database, entry

CCDS2593.1

(http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS2593)

CRELD1 Amplicon

Exons covered Genomic coordinates of amplicon boundaries

Amplicon size (bp)

A 5′UTR 9975524-9976133 609 B Exons 1- 2 9976078-9976670 592 C Exon 3 9979181-9979400 219 D Exon 4 9979619-9979946 327 E Exons 5-6 9982495-9982928 433 F Exons 7-9 9984441-9985250 809 G Exon 9b 9985625-9985935 310 H Exon 10-3′UTR 9986000-9986353 353


HEY2 was amplified by PCR from genomic DNA as 4 amplicons that span the 5 exons of the

gene, and includes intron-exon boundaries and at least 50 bp into each intron from the 5′ and 3′

boundaries and untranslated regions (UTR). Genomic coordinates are from the National Center

for Biotechnology Information Consensus Coding Sequence Database, entry CCDS5131.1

(http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS5131)

HEY2 Amplicon

Exons covered Genomic coordinates of amplicon boundaries

Amplicon size (bp)

A 5′UTR-exon 1 126079734-126071055 321 B Exon 2-3 126072767-126073376 609 C Exon 4 126075495-126075815 320 D Exon 5-3′UTR 126080138-126081072 934

http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS2593�

http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&DATA=CCDS5131�

CIRCCVG/2011/960872 /R2


Genotype frequencies are skewed in the Ts65Dn x Hey2+/- cross.

Genotypes Crosses

Total number Number of pups for each genotype/Percentage of total (%)

Ts65Dn x Creld1+/- 139

Eu, Creld1+/+ Eu, Creld1+/- Ts, Creld1+/+ Ts, Creld1+/-

52(37.4%) 37(26.6%) 25(18%) 25(18%)

Ts65Dn x Hey2+/- 182 Eu, Hey2+/+ Eu, Hey2+/- Ts, Hey2+/+ Ts, Hey2+/-

65(35.7%) 40(22%) 48(26.4%) 29(15.9%)*

Creld1+/- x Hey2+/- 112 Creld1+/+, Hey2+/+ Creld1+/-,

Hey2+/+ Creld1+/+,

Hey2+/- Creld1+/-, Hey2+/-

24(21.4%) 30(26.8%) 26(23.2%) 32(26.6%)

Creld1+/-,Hey2+/- x Hey2+/- 206

Creld1+/+, Hey2+/+ Creld1+/-, Hey2+/+

Creld1+/+, Hey2+/-

Creld1+/-, Hey2+/-

35(17%) 37(18%) 51(24.8%) 48(23.3%)

Creld1+/+, Hey2-/- Creld1+/-, Hey2-/-

20(9.7%) 15(7.2%)

* indicates significant difference between the observed and expected frequencies, p=0.04

(Fisher’s exact test, two-tailed).

CIRCCVG/2011/960872 /R3

Supplemental Fig 1. The RNA expression of Creld1 in mouse embryos with different gene

dosage. A) PCR used to genotype embryos from a Creld1+/- x Creld1+/- cross. Primer “KO-F”

was designed 5’ of the neo-cassestte and paired with a “Reverse” primer (designed in the 3’

homology region of the knockout cassette) to detect a 700 bp Creld1-knockout-allele amplicon.

A “WT-F” primer was made to bind within exon1 of Creld1 and also paired with the “Reverse”

primer to produce an amplicon of 528 bp, corresponding to the wildtype genotype. KO-F primer:

5’-CCAGTCAAAAACCACAGAGAGGG-3’, WT-F primer: 5’-CATCCTTCTCCCCGAGCTGAG-3’,

Reverse primer: 5’-GTGTTTCCACCCCCGAAGT-3’; B) The RNA expression of Creld1 in WT,

Creld1+/-, Creld1+/-,Hey2+/- and Creld1-/- embryos at E10.5, β-Actin was used as a loading control.

The primers for Creld1 PCR were: forward primer: 5’-ACCATGGCCCCATGGCCCCCGAA-3’,

reverse primer: 5’-TTACTTGTCATCGTCATCCTTGTAATCCTCGTCTTCTGTCATCT-3’.

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