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  Persistant ductus arteriosus (PDA) and coarctation of the aorta (CoA) are two
frequent forms of congenital heart disease which sometimes co-occur. A genetic basis for these
diseases has been suspected based on several reports of familial recurrence. We recently enrolled
an extended family with PDA and CoA. The presence of five inbreeding loops with 5 affected
individuals in consanguineous marriages strongly suggested autosomal recessive inheritance.
| 

Genomic DNA for linkage studies was isolated from peripheral blood lymphocytes using the
Gentra Puregene kit according to manufacturer protocol. Genotyping was performed the Infinium
HumanLinkage-12 chip (6090 SNPs). Logarithm of the odds (LOD) scores were determined by
two-point and multipoint linkage analysis using the FASTLINKv4.1 and Genehunter modules of
easyLINKAGEv2.10 assuming a recessive mode of inheritance. Additionally, a parametric two-
locus multipoint linkage analysis was performed using Superlink.
We obtain highly significant non-parametric LOD scores of 4.97 and 6.59 on chromosomes 9 and
16, respectively. Further refinement of this scenario using a parametric two-locus model shows a
highly significant LOD score of 5.3 for a digenic model. Haplotype analyses defines two candidate
intervals which are 8.9Mb and 1.2MB in size and harbour a total of 290 genes, many of which are
plausible biological candidates.
 
We report the first recessive family with PDA/CoA which is amenable to genetic mapping. Our
results show significant linkage of PDA/CoA to chromosome 9 and 16. To our knowledge, this is
the first digenic model of inheritance for a congenital heart disease. Since homozygosity at a
single locus is insufficient for the development of disease, epistasis at two loci is required for full
disease penetrance.
 @
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„ Ductus arteriosus (DA) is a left-to-right arterial shunt connecting the

pulmonary artery and the aortic arch. In utero, it allows the blood from the
right ventricle to bypass the fetal pulmonary circulation.
„ Patent DA arises when the DA fails to close postnatally, leaving a patent
connection between systemic and pulmonary circulation
„ Depending on the size of the PDA, the gestational age of the neonate, and
the pulmonary vascular resistance, a premature neonate may develop life-
threatening pulmonary overcirculation in the first few days of life.
 

„ Coarctation of the aorta (CoA) is a constricted aortic segment that

comprises localized medial thickening, with some infolding of the medial
and superimposed neointimal tissue.
„ CoA of the aorta may occur as an isolated defect or in association with
various other lesions like PDA.
„ CoA can often be diagnosed only after closure of the DA. Constriction of
the aorta by juxtaductal tissue is a proposed mechanism.
 

„ The study was approved by the Institutional Review Board (IRB) and
informed consent was obtained from all participants.
„@ 
„ All family members were evaluated by clinical history and physical
examination. Twelve-lead ECG and transthoracic 2D-
echocardiography were obtained.
„ !"|#


„ Genomic DNA for linkage studies was isolated from peripheral blood
lymphocytes using the Gentra Puregene kit.
„ Genotyping was performed using the 6090 SNPs Infinium
HumanLinkage-12 Genotyping BeadChip
„ ALOHOMORA and PedCheck was used for quality control
„!"|#


„ LOD scores were determined by two-point and multipoint linkage
analysis using easyLINKAGEv2.10. Two-Locus multipoint linkage
analysis was performed using SuperLink.
 |$%&#'@||

„ This family is characterized by the presence of five inbreeding loops with

five affected in consanguineous marriages
„ Inbreeding loops are highlighted by red boxes
„ Individuals 60 and 74 are depicted twice to facilitate graphical
representation
„ This family is compatible with autosomal recessive mode of inheritance
 # @&!"|#



„ Multipoint parametric analysis fails to reach significant linkage across the

whole genome
„ This result suggests that PDA and CoA do not segregate in a classical
monogenic Mendelian fashion
 #  (@&!"|#



„ The non-parametric whole genome analysis yields 4 significant linkage

peaks on chromosome 2,8,9 and 16.
„ Haplotype analysis showed that chromosome 2 and 8 loci are either :
„ not shared among affected individuals
„ shared by affected and unaffected individuals
"# @&!"|#


 
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„ A Two-Locu (digenic) multipoint parametric linkage analy i how a

ignificant linkage peak (max LOD . ) at marker r and
r in a rece ive-rece ive linkage matrix
„ Additive, multiplicative and heterogenou matrice did not reach
ignificance
„ The ignificance cut-off for a digenic linkage cenario i .
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„ 5 affected and 1 unaffected are homozygous

„ 2 unaffected are heterozygous
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„ The interval pinpointed by the digenic model yields 290 positional
candidates over 24.7Mb
„ Biological prioritization was performed to guide PCR amplification and
direct sequencing
„ Candidates were selected in their plausible or know role in normal ductal
closure
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 323.  Important role in cel-to-cell adhesion.
CDH5 is the vascular cadherin.
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Direct sequencing of the selected biological candidates failed to pinpoint a
potential deleterious mutation except CDH5.

Sequencing of CDH5 reveals :

a.) Homozygosity (C/C) for a very rare non-synonymous SNP (rs1965604)
b.) Homozygosity for a frequent non-synonymous SNP (rs1049970) in close
proximity (42bp)
c.) Homozygosity for this SNP in affected and unaffected individuals.

Pt. #
1

Pherogram showing rs1965604 in 2

Genotype frequencies for rs1965604 affected and 2 unaffected individuals
 $( 5"
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muality Control Steps
1 A ilent

Step 1 Andelfiner

Analysis of Sheared
DNAs

Step 2
3
Analysis of prepped 2
DNAs 1

Step 3 1 2 3

Analysis of captured
DNAs
„ The histo ram shows yield in Fluorescent Unit (FU) at each step of exome
capture as per Bioanalyzer 21 quality control
„ Our capture quality and yield is superior to manufacturer expectation for
step 2 and 3
 




„ First study to identify a two-locus model of inheritance for a CHD

„ This model support evidence for gene-gene interactions in the pathogenesis
of PDA and CoA
± Recessive-recessice matrix yields significant LOD scores
± Additive, multiplicative and heterogenous matrices were tested and failed to yield
significant LOD scores
„ First succesful mapping effort of PDA and CoA
„ Compatible with insight from animal models:
± Double homozygosity required for full penetrance of phenotype in several mouse
models
„ The critical intervals contains numerous biologically plausible candidate
genes.
± Direct sequencing of prioritized gene shows suggestive evidence for a role
of a rare allele in CDH5 conferring susceptibility to PDA and CoA
„ Identification of causative genes may help to identify novel pharmaceutical
targets to modulate ductal patency
„ Whole-exome sequencing in process to accelerate mutation discovery
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