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Chapter Title Essential in Genetic Etiology of Congenital Heart Diseases
Copyright Year 2018
Copyright Holder Springer International Publishing AG
Author Family Name Jinga
Particle
Given Name Mariana
SuffixDivision
Organization/University “Carol Davila” University of
Medicine and Pharmacy
Address Bucharest, Romania
Division
Organization/University “Carol Davila” Central Military
Emergency University Hospital
Address Bucharest, Romania
Author Family Name Dumitrescu
Particle
Given Name Silviu
Suffix
Division Department of Interventional
Cardiology
Organization/University “Carol Davila” Central Military Emergency University Hospital
Address Bucharest, Romania
Author Family Name Stan
Particle
Given Name Liviu
SuffixDivision Department of Cardiovascular
Surgery
Organization/University Central Clinic Emergency Military Hospital “Carol Davila”
Address Bucharest, RomaniaQ1

Author Family Name Bontas
Particle
Given Name Ecaterina
Suffix
Division Department of Cardiology
Organization/University “Carol Davila” Central Military
Emergency University Hospital
Address Bucharest, Romania
Author Family Name Paduraru
Particle
Given Name Tudor
Suffix
Division Department of Anesthesiology
Organization/University “Carol Davila” Central Military
Emergency University Hospital
Address Bucharest, Romania
Corresponding Author Family Name Tintoiu
Particle
Given Name Ion C.
Suffix
Division Department of Interventional
Cardiology
Organization/University “Carol Davila” Central Military
Emergency University Hospital
Address Bucharest, Romania
Author Family Name Murgu
Particle
Given Name Vasile
Suffix
Division Faculty of Medicine
Organization/University “Titu Maiorescu” University
Address Bucharest, Romania
Author Family Name Zoabi
Particle El
Given Name Rabia Denis
Suffix

Division Faculty of Medicine
Organization/University “Titu Maiorescu” University
Address Bucharest, Romania
Abstract Congenital heart disease (CHD) represent another unsolved problem of the
present, although new techniques of exploration like fluorescence in situ
hybridization (FISH), high resolution array-comparative genomic hybridization
(array-CGH), single nucleotide polymorphisms (SNPs), comparative genomic
hybridization (CGH) and spectral karyotyping (SKY) explain some genetic
mechanisms implied in the genesis of this pathology. However, there are many
unresolved issues. Structural modifications of chromosomes by duplication
and also by deletion determine variable phenotypes depending on the altered
structural site. Given that, the genetic defect affects only one gene or more but
determines repercussions over the cardiac anatomy or/and other non-cardiac
systems, as a result the phenotype can be syndromic or nonsyndromic. The raised
or low number of chromosomes was the first explanation for CHD. Trisomy
21 (Down’s syndrome), 18 (Edward’s syndrome), 13 (Patau’s syndrome),
monosomy X (Turner’s syndrome), deletion at chromosome 7q11.23 and
22q11.2, multiple gene mutations, are the most frequent chromosomal or sub-
chromosomal destructuring along with syndromic phenotype. Copy number
variations (CNVs) is defined as sub-chromosomal mechanism that by deletion
or multiplication produces in general nonsyndromic phenotype. Transcription
factors T-Box protein 5 (TBX5), mNK2 Homeobox 5 (NKX2.5), GATA- binding
protein 4 (GATA4) are specific proteins that sending information from DNA to
RNA messenger can undergo structural denaturations and if this happens these
proteins cannot send correct messages resulting in alteration of the normal
chain of cardiogenesis. Combination between these mechanisms and unitary
action produce CHD in different forms, syndromic or nonsyndromic.
Keywords (separated
by “ – ”)Chromosome abnormality – Gene defects – Syndromic – Nonsyndromic –
Duplication – Deletion – Congenital heart diseases – Developmental delay
AUTHOR QUERIES
Q1 Please check if the affiliations are presented correctly.

© Springer International Publishing AG 2018
S. I. Dumitrescu et al. (eds.), Right Heart Pathology , https://doi.org/10.1007/978-3-319-73764-5_13Essential in Genetic Etiology
of Congenital Heart Diseases
Mariana Jinga, Silviu Dumitrescu, Liviu Stan,
Ecaterina Bontas, Tudor Paduraru, Ion C. Tintoiu,
Vasile Murgu, and Rabia Denis El Zoabi
Abstract
Congenital heart disease (CHD) represent
another unsolved problem of the present,
although new techniques of exploration like
fluorescence in situ hybridization (FISH), high
resolution array-comparative genomic hybrid –
ization (array-CGH), single nucleotide poly –
morphisms (SNPs), comparative genomic
hybridization (CGH) and spectral karyotyping (SKY) explain some genetic mechanisms
implied in the genesis of this pathology.
However, there are many unresolved issues.
Structural modifications of chromosomes by
duplication and also by deletion determine
variable phenotypes depending on the altered
structural site. Given that, the genetic defect
affects only one gene or more but determines
repercussions over the cardiac anatomy or/and
other non-cardiac systems, as a result the phe –
notype can be syndromic or nonsyndromic.
The raised or low number of chromosomes
was the first explanation for CHD. Trisomy 21
(Down’s syndrome), 18 (Edward’s syndrome),
13 (Patau’s syndrome), monosomy X (Turner’s
syndrome), deletion at chromosome 7q11.23
and 22q11.2, multiple gene mutations, are the
most frequent chromosomal or sub-chromo –
somal destructuring along with syndromic
phenotype. Copy number variations (CNVs) is
defined as sub-chromosomal mechanism that
by deletion or multiplication produces in gen –
eral nonsyndromic phenotype. Transcription
factors T-Box protein 5 (TBX5), mNK2
Homeobox 5 (NKX2.5), GATA- binding pro –
tein 4 (GATA4) are specific proteins that send –
ing information from DNA to RNA messenger
can undergo structural denaturations and if this
happens these proteins cannot send correct
messages resulting in alteration of the normal
chain of cardiogenesis. Combination between M. Jinga
“Carol Davila” University of Medicine and
Pharmacy, Bucharest, Romania
“Carol Davila” Central Military Emergency
University Hospital, Bucharest, Romania
S. Dumitrescu · I. C. Tintoiu ( *)
Department of Interventional Cardiology, “Carol
Davila” Central Military Emergency University
Hospital, Bucharest, Romania
L. Stan
Department of Cardiovascular Surgery, Central Clinic
Emergency Military Hospital “Carol Davila”,
Bucharest, Romania
E. Bontas
Department of Cardiology, “Carol Davila” Central
Military Emergency University Hospital,
Bucharest, Romania
T. Paduraru
Department of Anesthesiology, “Carol Davila”
Central Military Emergency University Hospital,
Bucharest, Romania
V . Murgu · R. D. El Zoabi
Faculty of Medicine, “Titu Maiorescu” University,
Bucharest, RomaniaAU1131
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these mechanisms and unitary action produce
CHD in different forms, syndromic or
nonsyndromic.
Keywords
Chromosome abnormality · Gene defects ·
Syndromic · Nonsyndromic · Duplication ·
Deletion · Congenital heart diseases ·
Developmental delay
13.1 Introduction
CHDs represent the single largest cause of infant
morbidity and mortality worldwide [ 1, 2], and
CHD genetics is increasing with a fast rate [ 3].
However, CHDs continue to have unresolved
issues because of multiple aetiologies forms such
as chromosome abnormalities caused by envi –
ronmental factors and genetic disorders; single
or multiple gene mutations; de novo mutations;
abnormal RNA; single nucleotide polymorphism;
copy number variations (CNVs).
Mechanisms of genesis and understanding
of congenital cardiac malformations have con –
cerned scientific societies over two decades.
Although genetic elucidation of cardiogenesis
enlightened some of the issues, there are still
many unknown mechanisms that presently are in
stage of hypothesis. However the progression of
understanding the involvements of molecular and
genetic mechanisms which trigger the defects in
cardiac structure was progressive mainly with the
help of new modern techniques used in studies of
this pathology (Table 13.1) [4].
Issy-Les-Moulineaux, 22-Feb-2017
To the attention of Dr. Ecaterina Bontas
Dear,
As per your request below, we hereby grant you
permission to reproduce the material detailed in
your request in print and electronic formats at
no charge subject to the following conditions:
If any part of the material to be used (for
example, figures) has appeared in our
publication with credit or acknowledgement to another source, permission must also be
sought from that source. If such permission is
not obtained then that materials may not be
included in your publication.
Any modification of the material is likely to
harm the moral right of the authors and
therefore should be first submitted and
approved by the authors who are the sole
owner of the moral right.Table 13.1 Conceptual evolution of the genetics of con –
genital heart diseases
Concepts Examples
Multifactorial
inheritanceAll heart diseases
Major role of the
environmentTeratogenic: Rubella,
thalidomide
Unique mechanism of
anatomically different
heart disease: One
genetic abnormality–
several heart diseasesDeletion of chromosome
22q1.1 and conotruncal heart
diseases
Monogenic nature of
many heart diseasesInteratrial communication,
atrioventricular canals,
tetralogy of Fallot
Failure of strategies of
partial phenocopy:
Genetically different
syndrome and
non-syndrome
associated heart
diseasesInteratrial communication
and Holt-Oram syndrome
(TBX5 ), tetralogy of Fallot
and deletion of chromosome
22q1.1, atrioventricular
canals and critical cardiac
region of trisomy 21
Notion of phenotype
continuum or gravity
spectrumBicuspid aortic valve, aortic
stenosis and coarctation,
shone syndrome, hypoplasia
of left heart
Variability of
intrafamilial
expression for a same
molecular abnormalityFamilial heart diseases of
deletion of chromosome
22q1.1
Genetic heterogeneity
of congenital heart
diseases: One
malformation–several
genesInteratrial communication
and mutations in NKX2.5 ,
GATA4 , MYH7
Heterogeneity of
mechanisms for a
same heart diseaseCommon arterial trunk:
Septation disease of the
efferent pathway or of
myocardium rotation from
the base of the efferent
pathway
Redefinition of the
phenotype in relation
to the mechanismDouble outlet right ventricles
From Bajolle et al. [4] with permission
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Senior Copyrights Coordinator,
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ELSEVIER | Global Book Production
13.2 Causes of Congenital Heart
Defects
New concepts regarding the etiology of CHD
sustain genetic and nongenetic factors. Among
genetic factors the most important are structural
alteration of chromosomes responsible for car -diogenesis, genetic mutations (8%), restructur –
ing of RNA, epigenetic factors, single nucleotide
polymorphism and multifactorial factors (90%)
(Fig. 13.1) [5].
It has to be mentioned that James Nora suggested
in 1968 the contribution of combining genetic and
environmental factors in the development of a
human heart defects that can be used in prevention
of CHD [ 6, 7]. Recently, Akhirome et al. [6] showed
in a comprehensive review that genetic etiologies of
CHD comprise an high number of unknown causes
(61.7%), the rest being known (Table 13.2) [6].
Importantly, the known causes of CHD are de novo
or inherited genetic abnormalities [ 6].
13.3 Chromosomal Abnormalities
Chromosomal aneuploidy is the most frequent
cause of chromosome destructuring that modi –
fies the normal genetic information. Due to the etiology of CHDrubella
teratogens
maternal diseases
Altered haemodynamics
abnormal chromosome structure
gene mutations
abnormal RNA
epigenetics
single nucleotide polymorphismsenvironmental factors
genetic factorsFig. 13.1 Etiology of
congenital heart defects
[5]. It is open access
chapter . Attribution 3.0
Unported (CC BY 3.0)AU4
Table 13.2 Genetic disorders in congenital heart defects
(adapted from Akhirome et al. [6])
Genetic disorder % References
Chromosomal syndrome 12 [8, 9]
De novo copy number variations
(CNV)15 [10]
De novo gene mutation 10 [11–13]
Inherited gene mutation 1.3 [13]
Unknown 61.7 [6]
13 Essential in Genetic Etiology of Congenital Heart Diseases96
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new techniques of study like fluorescence in
situ hybridization—FISH, spectral karyotyp –
ing (SKY) and comparative genomic hybrid –
ization (CGH), it appeared new concepts as
cells genetics (molecular cytogenetics) in the
last decades. These new concepts established
that deletion and duplication (microdele –
tions and microduplications) are new genetic
mechanisms of chromosome destructuring in
CHD [ 14].
Genetic modifications that affect only the heart
are classified in the nonsyndromic CHD category,
however those that involve heart and other organs are named syndromic CHD. Recently, [ 15] clas –
sified by a study the chromosomal anomalies
from CHD into three categories [ 15]:
1. Modification of chromosomes implied in car –
diogenesis such as trisomy 21 (Down’s syn –
drome), 18 (Edward’s syndrome), 13 (Patau’s
syndrome), monosomy X (Turner’s syn –
drome), Tetrasomy 22q [ 15];
2. Chromosomes deletion: 22q11, 7q11.23,
1p36, and 22q11.2;
3. Gene mutations: multiple, (TFAP2B), or sin –
gle (TFAP2B) (Table 13.3) [15].
Table 13.3 Representative chromosomal disorders associated with congenital heart defects [ 16] with permission
Chromosomal disorder Main featuresPercent
with
CHD Heart anomaly References
Deletion 4p
(Wolf- Hirschhorn
syndrome)Pronounced microcephaly, widely spaced
eyes, broad nasal bridge (Greek helmet
appearance), downturned mouth,
micrognathia, preauricular skin tags,
elongated trunk and fingers, severe mental
retardation and seizures; 1/3 die in infancy50–65 ASD, VSD, PDA,
LSVC, aortic atresia,
dextrocardia, TOF,
tricuspid atresia[17, 18]
Deletion 5p
(cri-du-chat)Catlike cry, prenatal and postnatal growth
retardation, round face, widely spaced
eyes, epicanthal fold, simian crease,
severe mental retardation, long survival30–60 VSD, ASD, PDA [17, 19,
20]
Deletion 7q11.23
(Williams-Beuren
syndrome)Infantile hypercalcemia, skeletal and renal
anomalies, cognitive deficits, “social”
personality, elfin facies53–85 Supravalvar AS and
PS, PPS[21–23]
Trisomy 8 mosaicism Skeletal/vertebral anomalies, widely
spaced eyes, broad nasal bridge, small
jaw, high arched palate, cryptorchidism,
renal anomalies (50%), long survival25 VSD, PDA, CoA, PS,
TAPVR, truncus
arteriosus[17,
24–27]
Deletion 8p syndrome Microcephaly, growth retardation, mental
retardation, deep-set eyes, malformed
ears, small chin, genital anomalies in
males, long survival50–75 A VSD, PS, VSD, TOF [28–30]
Trisomy 9 Severe prenatal and postnatal growth
retardation, marked microcephaly,
deep-set eyes, low-set ears, severe mental
retardation; 2/3 die in infancy65–80 PDA, LSVC, VSD,
TOF/PA, DORV[17, 31]
Deletion 10p Frontal bossing, short down-slanting
palpebral fissures, small low-set ears,
micrognathia, cleft palate, short neck,
urinary/genital, upper-limb anomalies50 BA V , ASD, VSD,
PDA, PS, CoA,
truncus arteriosus[17, 32,
33]
Deletion 11q
(Jacobsen syndrome)Growth retardation, developmental delay,
mental retardation, thrombocytopenia,
platelet dysfunction, widely spaced eyes,
strabismus, broad nasal bridge, thin upper
lip, prominent forehead56 HLHS, valvar AS,
VSD, CoA, Shone’s
complex[34]AU2
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Chromosomal disorder Main featuresPercent
with
CHD Heart anomaly References
Trisomy 13 (Patau
syndrome)Polydactyly, cleft lip and palate, scalp
defects, hypotelorism, microphthalmia or
anophthalmia, colobomata of irides,
holoprosencephaly, microcephaly,
deafness, profound mental retardation, rib
abnormalities, omphalocele, renal
abnormalities, hypospadias,
cryptorchidism, uterine abnormalities;
80% die in first year80 ASD, VSD, PDA,
HLHS, laterality
defects, atrial
isomerism[35, 36]
Trisomy 18 (Edwards
syndrome)IUGR, polyhydramnios, micrognathia,
short sternum, hypertonia, rocker-bottom
feet, overlapping fingers and toes, TEF,
CDH, omphalocele, renal anomalies,
biliary atresia, profound mental
retardation; 90% die in first year90–100 ASD, VSD, PDA,
TOF, DORV , D-TGA,
CoA, BA V , BPV ,
polyvalvular nodular
dysplasia[17, 37,
38]
Deletion 20p12
(Alagille syndrome)Bile duct paucity, cholestasis, skeletal or
ocular anomalies, broad forehead, widely
spaced eyes, underdeveloped mandible85–94 Peripheral PA,
hypoplasia, TOF, PS,
(left-sided heart
lesions and septal
defects less common)[39, 40]
Trisomy 21 (Down
syndrome)Hypotonia, hyperextensibility, epicanthal
fold, simian crease, clinodactyly of fifth
finger, brachydactyly, variable mental
retardation, premature aging40–50 A VSD, VSD, ASD,
(TOF, D-TGA less
common)[17,
41–46]
Deletion 22q11
(DiGeorge,
velocardiofacial, and
conotruncal anomaly
face syndrome)Hypertelorism, micrognathia, low-set
posteriorly rotated ears, “fish mouth,”
thymic and parathyroid hypoplasia,
hypocalcemia, feeding/speech/learning/
behavioral disorders, immunodeficiency,
palate/skeletal/renal anomalies75 IAA-B, truncus
arteriosus, isolated
aortic arch anomalies,
TOF, conoventricular
VSD[47, 48]
Monosomy X (turner
syndrome, 45,X)Lymphedema of hands and feet, widely
spaced hypoplastic nipples, webbed neck,
primary amenorrhea, short stature, normal
intelligence25–35 CoA, BA V , valvar AS,
HLHS, aortic
dissection[17,
49–53]
Klinefelter syndrome
(47,XXY)Usually normal appearing, tall stature,
small testes, delayed puberty, emotional
and behavioral problems common,
variable mental retardation50 MVP, venous
thromboembolic
disease, PDA, ASD[17, 54]
Abbreviations: CHD indicates congenital heart defects, ASD atrial septal defect, VSD ventricular septal defect, PDA
patent ductus arteriosus, LSVC persistent left superior vena cava, TOF tetralogy of Fallot, AS aortic stenosis, PS pul-
monic stenosis, PPS peripheral pulmonary stenosis, CoA coarctation of the aorta, TAPVR total anomalous pulmonary
venous return, AVSD atrioventricular septal defect, TOF/P A tetralogy of Fallot with pulmonary atresia, DORV double-
outlet right ventricle, BAV bicuspid aortic valve, HLHS hypoplastic left heart syndrome, IUGR intrauterine growth
retardation, TEF tracheoesophageal fistula, CDH congenital diaphragmatic hernia, D-TGA D-transposition of the
great arteries, BPV bicuspid pulmonary valve, PA pulmonary artery, IAA-B interrupted aortic arch type B, and MVP
mitral valve prolapseTable 13.3 (continued)
Moreover, the most frequent congenital mal –
formations with modification of chromosomes
implied in cardiogenesis are: atrial septal defect
(ASD) with all its types (ostium primum, ostium
secundum, and common atrioventricular canal), ventricular septal defect, and patent ductus arte –
riosus (PDA). For instance, trisomy 21 (Down’s
syndrome) has a frequency of 40–50% from
cases [ 55], and 1 from 2500 female newborns
have monosomy X (Turner’s Syndrome) [ 56].
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Importantly, heart defects are present in 35% cases
of trisomy 13 (Patau’s syndrome) and in 45%
cases of trisomy 18 (Edward’s syndrome) [ 57].
Also, the known genetic mechanisms that are
assigned to chromosome modifications implied
in cardiogenesis or chromosome deletion are
present in Table 13.3 [16].
13.4 Copy Number Variations
(CNVs)
According to current evidence, along with
modification of chromosomes, microdeletion
and genetic mutations, another mechanism may
induce sub-chromosomal changes in genome
structure known as Copy Number Variations
(CNVs). These sub-chromosomal changes or
destructuring have outcomes over cardiogen –
esis and it is accomplished by multiple mecha –
nisms but mainly by deletions and duplications
(Fig. 13.2) [60].
In particular, investigations such as fluores –
cence in situ hybridization (FISH), high- resolution
array-comparative genomic hybridization (array-
CGH) and single nucleotide polymorphisms
(SNPs) are the most frequent methods used for
identification of CNVs and their connection to
CHD [ 60].
Richards et al. [61] on a study of 40 childrens
with CHD using fluorescence in situ hybridiza –
tion (FISH) consider that this technique is more
precisely in detection of modifications induced
by deletions, duplications or translocations,
especially in subthelomeric analyzing explaining
fundamental chromosomal and subchromosomal
mechanisms in genesis of CHD (Fig. 13.3) [61].
Fahed et al. [62] evaluated recently in a meta –
nalysis the variation of CNVs based on the number
and type of genes involved in the developmen –
tal of a pathological phenotype in patients with
non-syndromic CHD. The alteration mechanism
of CNVs is either the deletion or duplication. The
number of involved genes can be one or multiple
and their destructuring generate various types of
CHD. For instance the most frequent modified
genes in genesis of Fallot tetralogy are only one
(PPM1K, MID1 ) or multiple ( RAF J, TMEM40, EDIL3, VCAN, SSBP2, TMEM167A, CNOT6,
GFPT2, FLT4, ZNF879, ZNF345C, ADAMTS2,
NSD1, GATA4, NEIL2, FDFT1, CSTB, SOX7,
NOTCH1, EHMT1, TNFSF11, MIER2, CNN2,
FSTL3, PTBP1, WDR18, GNA11, S1PR4 ), that
further causes de novo CNVs responsible for the
genesis of Fallot tetralogy, when while both par –
ents have normal karyotype. Also, this metaanal –
ysis showed that 5–10% of patients are estimated
to have non- syndromic CHD while both parents
have normal karyotype (see Table 13.4) [62].
13.5 Transcription Factors
Transcription factors (sequence-specific DNA-
binding factor) are specific proteins with the role
to control the transfer of genetic information
from DNA to RNA messenger [ 71]. This process
is accomplished through mechanisms of activa –
tion or blockage of RNA polymerase [ 72–74].
In the differentiation and embryological evo –
lution of cardiac structures with effects on ana –
tomical integrity the main role belongs to the
transcription factors which are T-Box protein
5 (TBX5 , NK2 Homeobox 5 ( NKX2.5 ), GATA-
binding protein 4 ( GATA4 ). They control tran –
scription of the information from DNA to RNA
messenger through RNA polymerase [ 71, 75].
TBX5 , GATA4 , and NKX2 are the main factors
of transcription that control the differentiation
and development of cardiac structures and they
are responsible for the congenital malformations.
These can relate each other or they can act alone
in the genesis of CHD. Thus, in case of ASD and
VSD, each of them can be implied in the devel –
opment of congenital malformation. For ASVD,
both transcription factors like TBX5 and GATA4
can be involved. The specificity of one transcrip –
tion factor exists in Ebstein disease ( NKX2-5 )
and hypoplasic left ventricle; for paroxistic
atrial fibrillation and Holt-Oram Syndrome is
responsible only TBX5 . Further, for Fallot tetral –
ogy is implied only NKX2-5, and for A V block
the same NKX2-5 . Also one transcription factor
is responsible for the pulmonary valve stenosis
known as GATA4 . Combinations between these
major transcription factors participate to genesis
M. Jinga et al.173
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a
bChromosome
Pair
CNVs
Tandem
Duplication
Deletion
Contiguous gene disruption
Whole gene
(or partial intergenic) deletion
Unmasking of a recessive allele
(Compound heterozygosity)
Disruption of regulatory element(s)Diploid
Fig. 13.2 Examples of CNV and associated disease
mechanisms. ( a) Normal diploid status and two exam –
ples of CNV (a simple deletion and a tandem duplica –
tion). Not pictured are the diverse other forms of CNV ,
including non-contiguous insertions, higher-order copy
number changes (multi-allelic CNV), and more com –
plex rearrangements. CNVs may involve no, one or
multiple genomic elements. ( b) Selected mechanisms
underlying disease effects of copy number losses (dele –
tions). A gene is indicated by a contiguous monochro –
matic set of rectangles, and a regulatory element (e.g., promoter) by an oval. The definition of ‘gene’ extends
beyond protein- coding genes to potentially include non –
coding elements like microRNAs and long noncoding
RNAs. Of note, duplications can effect change through
increased copy number of a dosage sensitive gene (not
pictured) or via the mechanisms depicted for deletions
(e.g., via disruption at a breakpoint or partial intragenic
duplication). Inspired by Fig. 1 in Bassett et al. [58] and
Fig. 2 in Lee and Scherer [ 59]. From Costain et al. [60].
It is an open access article . Attribution 4.0 International
(CC BY 4.0)AU5
13 Essential in Genetic Etiology of Congenital Heart Diseases

ab1
c1 c2 db2
Fig. 13.3 FISH demonstrates chromosomal abnormali –
ties in five subjects with CHD and additional anomalies.
(A) Interstitial duplication of long arm of chromosome
(ch) 2. FISH using custom BAC clones shows normal
hybridization signals to the normal homologue of ch2
(arrowhead ) and duplicated hybridization signals to the
abnormal homologue of ch2 ( arrow ). Hybridization sig –
nals are also seen in interphase cells (right) with long
arrows showing two signals (duplication) and arrowhead
showing a single signal (normal). ( B) Unbalanced translo –
cation involving the long arm of chromosome 16 and
short arm of ch19. ( B1) FISH using subtelomeric probes
to the short arm ( green signal ) and long arm of ch16 ( red
signal ) indicate trisomy for the terminal region of ch16.
(B2) FISH using probes for the subtelomeres of the short
arm ( green ), long arm ( red), and centromere (aqua) of
ch19. Absence of the green signal ( arrowhead ) indicative
of deletion of the distal segment of the short arm of ch19
when compared with normal ch19 (arrow). ( C)
Unbalanced translocation involving the long arm of ch1
and the long arm of ch15. ( C1) FISH using subtelomeric
sequences for the short arm ( green ) and the long arm (red)
of chromosome 1. Arrow identifies the distal long arm of
the abnormal chromosome 1 (signal missing), arrowhead
identifies the distal long arm of the normal chromosome 1.
Additional signals ( yellow ) in C1 identify Xp/Yp subtelo -meric regions used as reporter sequences. ( C2) Arrows
identify hybridization signals for the subtelomeric
sequences of ch15. Arrowhead indicates a ch15q hybrid –
ization signal on the long arm of ch1. Additional signals
in C2 indicate short arm of ch10 ( green ) and long arm of
10 (red) as reporter sequences. ( D) FISH showing normal
hybridization to the DiGeorge/velo-cardio-facial syn –
drome critical region at chromosome 22q11.2 using a
TUPLE1 probe. Arrowhead identifies normal hybridiza –
tion pattern ( red), arrow points to the deleted region.
Green signal identifies distal ch22q, a reporter sequence
encoding the arylsulfatase A gene. ( E) Unbalanced trans –
location involving the long arm of ch7 and the long arm of
ch17. ( E1) FISH showing hybridization of subtelomeric
sequences to the short arm ( red) and the long arm ( green )
of ch7. Arrows indicate hybridization to the long arms of
both the normal and abnormal homologues of ch7 indicat –
ing that the subtelomeric sequences on the abnormal chro –
mosome are intact. The second set of signals is a reporter
and identifies ch14. ( E2) Arrowhead identifies hybridiza –
tion to the telomeres of the long arms of the normal homo –
logues of ch17. Arrow identifies a ch17 hybridization
signal on the distal long arm of ch7. The scale bar in ( A)
represents 5 μm and the same magnification of 600× is
used in all images. From Richards et al. [ 61] with
permission
M. Jinga et al.

Table 13.4 Copy number variations (CNVs) associated with recurrent cases of non-syndromic CHD
LocusSize
range
(Kbp)No
of
cases Inheritance CNVsNo of
genes GenesaPhenotype Reference(s)
1q21.1 418–
398121 De novo,
inherited,
n/aGain,
loss3–45 PRKAB2, FM05,
CHD1L, BCL9,
ACP6, GJA5,
CD160, PDZK1,
NBPF11,
FMO5, GJA8TOF, AS, CoA,
PA, VSD[63–67]
3p25.1 175–
12,3803 De novo,
inheritedGain 2 RAF J,
TMEM40TOF [64, 68]
3q22.1–3q26.1 680–
32,1343 Inherited,
n/aGain,
loss0–300 FOXL2, NPHP3,
F AM62C,
CEP70, F AIM,
PIK3CB,
FOXL2,
BPESC1DORV , TAPVR,
A VSD[69, 67,
70]
4q22.1 45 2 De novo
inheritedGain 1 PPM1K TOF [64, 63]
5q14.1-q14.3 4937–
54542 De novo Gain 41,103 EDIL3, VCAN,
SSBP2,
TMEM167ATOF [63, 65]
5q35.3 264–
17774 De novo,
n/aGain 19–38 CNOT6, GFPT2,
FLT4, ZNF879,
ZNF345C,
ADAMTS2,
NSD1TOF [63, 67]
7q11.23 330–
3482 n/a Gain 5–8 FKBP6 HLHS, Ebstein’s [67]
8p23.1 67–
12,00010 n/a Gain,
loss4 GATA4, NEIL2,
FDFT1, CSTB,
SOX7A VSD, VSD,
TOF,ASD, BA V[63, 67]
9q34.3 190–
2633 De novo Loss 2–9 NOTCH1,
EHMT1TOF, CoA,
HLHS[63, 67]
11p15.5 256–
2712 n/a Gain 13 HRAS DILV , AS [67]
13q14.11 555–
14303 n/a, de
novoGain 7 TNFSF11 TOF, TAPVR,
VSD,BA V[65, 69]
(continued)
e1 e2
Fig. 13.3 (continued)
13 Essential in Genetic Etiology of Congenital Heart Diseases
t4.1
t4.2
t4.3
t4.4
t4.5
t4.6
t4.7
t4.8
t4.9
t4.10
t4.11
t4.12
t4.13
t4.14
t4.15
t4.16
t4.17
t4.18
t4.19
t4.20
t4.21
t4.22
t4.23
t4.24
t4.25
t4.26
t4.27
t4.28
t4.29
t4.30
t4.31
t4.32
t4.33
t4.34
t4.35
t4.36
t4.37
t4.38
t4.39

of cardiac structures, therefore TBX5 with NKX2-
5 control beginning with the first heart field
(FHF) stage cellular differentiation and later the
specific genes for cardiac cavities and conduc –
tion system of cardiac tissue. Moreover, GATA4
and NKX2-5 promote proteins from sarcomeric
genes, and GATA4 + TBX5 sustain gene expres –
sion for protein synthesis from A V node and gap-
junction (Fig. 13.4) [76].
To sum up, these hypotheses don’t clarify the
questions regarding why same gene modifica –
tions can develop more types of CHD, thus the
mutations of TBX5 can lead to ASD phenotype
and also to VSD, or mutations of TBX5 , TBX20 ,
GATA4 and NKX2.5 can produce different phe –
notype on a patient than to another patient [ 76].
Furthermore, they don’t explain why only one
mutation can decide a unique phenotype, for
example NKX2-5 for Fallot Tetralogy and GATA4
for pulmonary valve stenosis. However, Kloesel
et al. [76] considers that the explanation for these
situations are realized transcriptional/translation
and by interaction of multiple genes [ 76].13.6 Syndromic CHD
Precisely, genetic defects responsible for congen –
ital heart diseases are generating damaging of the
cardiac phenotype (non-syndromic CHD) or they
are accompanied by other extracardiac defects
(syndromic CHD).
Genetic mechanisms responsible for both
conditions are multiple, some of them in hypoth –
eses stage. For syndromic CHD there are genetic
mechanisms which include disorders of chromo –
somes number (chromosomal anueploidy), chro –
mosomal microdeletions, single gene defects,
gene mutations. As a result, specific genetic
defects are accomplished by mechanisms such as
deletion (22q11.2), microdeletion ( ELN, 20p12,
12q21, 2-q22), mutations [ ELN, TBX5 , JAG1 or
Notch1 , PTPN11 , SOS1 , RAF1 , KRAS , BRAF ,
MEK1 , MEK2 , and HRAS , CHD7 , SEMA3E ,
TF AP2B , EVC or EVC2 , HRAS , Fibrillin-1 ),
outlining a clinical picture specific for each
genetic defect. These mechanisms can act alone
or combined, as a consequence we have different Table 13.4 (continued)
LocusSize
range
(Kbp)No
of
cases Inheritance CNVsNo of
genes GenesaPhenotype Reference(s)
15q11.2 238–
228512 n/a Loss 4 TUBGCP5,
CYFIP1, NIP A2,
NIP A1CoA, ASD,
VSD, TAPVD,
complex
left-sided[63]
16p13.11 1414–
29033 n/a Gain 11–14 MYH11 Malformatio-ns
HLHS[67]
18q11.1–
18q11.2308–
61182 n/a Gain 1–28 GATA6 VSD [67]
19p13.3 52–
8053 n/a, de
novoGain,
loss1–34 MIER2, CNN2,
FSTL3, PTBP1,
WDR18,
GNA11, S1PR4TOF [65, 63]
Xp22.2 509–
6152 n/a Gain 2–4 MID1 TOF, A VSD [65]
From Fahed et al. [62] with permission
aGenes listed are encompassed by the CNV and were reported by the authors as candidate genes that are responsible for
CHD. Only CNVs that have recurred in ≥1 CHD patient are listed. CNVs copy number variants, AVSD atrioventricular
septal defects, ASD atrial septal defect, VSD ventricular septal defect, CoA coarctation of aorta, PS pulmonary stenosis,
TOF tetralogy of Fallot, BAV Bicuspid Aortic Valve, AS Aortic Stenosis, P APVR Partial Anomalous Pulmonary Venous
Return, TAPVR Total Anomalous Pulmonary Venous Return, TAPD Total Anomalous Pulmonary Venous Drenaige,
HLHS Hypoplastic left heart syndrome, DILV Double Inlet Left Ventricle, DORV Double outlet right ventricle
M. Jinga et al.
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266
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285286
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t4.41
t4.42
t4.43
t4.44
t4.45
t4.46
t4.47
t4.48
t4.49
t4.50
t4.51
t4.52
t4.53
t4.54
t4.55
t4.56
t4.57
t4.58
t4.59
t4.60

• Promotion of growth and differentiation of
posterior segment of heart and atrial and
LV precursors
• Formation of intraventricular septum
• Expression of gap-junction
protein in AV-node
• Activation of endothelial-to-mesenchymal
transformation
+ SMAD4: interaction is important for
cardiac septation• Endocardial cushion formation
• Regulates onset of cardiac differentiation
• Promotion of cardiac
sarcomeric protein gene
expression• Formation of intraventricular septum• Promotion of terminal differentiation of
myocardium
+ JARID2: regulates OFT development
+ SHOX2: inhibits NKx2-5 expression,
necessary for sinus node formation
+ MEF2C: establishes ventricular identity• Formation of intraventricular septum
• Expressed by cells of the SHF and PE• Expressed by cells of the FHF
• Control of heart chamber
development (activation of
chamber-specific genes, Nppa)
• Patterning of heart conduction
systemTBX5 + NKx2-5
GATA4 + TBX5
GATA4 + NKx2-5TBX5
NKx2-5Key Regulators
of Cardiac
Differentiation
GATA4ASD
VSD
AVSD
Paroxysmal AFIB
Holt-Oram syndrome
ASD ASD
VSD VSD
AVSD TOF
AV-block
Ebstein’s
anomaly
Hypoplastic left
heart syndromePulmonary
stenosis
Fig. 13.4 Condensed overview of the function and interaction between
three major transcription factors that are viewed as key regulators of car –
diac differentiation: T-Box protein 5 (TBX5), GATA-binding protein 4
(GATA4), and NK2 Homeobox 5 (NKX2.5). AFiB indicates atrial fibrilla -tion; ASD, atrial septal defect; A VSD, atrioventricular septal defect; FHF,
first heart field; OFT, outflow tract; PE, proepicardium; SHF, second heart
field; VSD, ventricular septal defect. From Kloesel et al. [76] with
permission
13 Essential in Genetic Etiology of Congenital Heart Diseases

clinical pictures depending on the type of the
genetic defect (Table 13.5) [77].
13.7 Non-syntromic CHD
Non-syndromic CHDs are the result of only one
modified gene that shapes its own structure in
an abnormal way, and that doesn’t respect the genetic code of cardiogenesis. NKX2.5 , GATA4 ,
MYH6 , BMPR2 , CRELD1 , ALK2 , NOTCH1
and PROSIT-240 are the most frequent involved
genes which can produce by themself cardiac
congenital malformations (Table 13.6) [78].
13.8 New Approach
There are still many unknowns in understanding
the gene dysfunction in congenital heart disease.
Presently, there is knowledge about 50 genes that
by mutations cause CHD. Recent studies estab –
lish a more precise relationship between genetic
mutations and CHD genesis [ 79].
Zaidi et al. [11] analyzed the mutations on
4169 genes and they found an excess of a new
protein in the group of heart-specific genes (high
heart expression ( HHE ) gene), establishing 8
gene mutations in patients with cardiac mal –
formations. These mutations were produced by
the modification of histone via methylation of
H3K4me (histone H3, lysine 4). This pathway
is recognized as responsible for gene mutations
MLL2 , KDM6A , and CHD7 [11].
Later, Homsy et al. [12] studied the genetic
differences between patients with syndromic
heart disease (S-CHD) and non-syndromic
CHD (NS-CHD) and suggested that de novo Table 13.5 Syndromes manifesting congenital heart dis –
ease and their genetic cause
Syndrome with CHD Genetic cause for CHD
Disorders of chromosome dosage
Trisomy 21 (Down
syndrome)Unknown
Turner Unknown
Chromosomal microdeletions
Di Georges syndrome 22q11.2 deletion resulting in
absent TBX1 gene
Williams-Beuren
syndromeMicrodeletion of ELN gene;
mutations in ELN gene
Single gene defects
Holt-Oram syndrome TBX5 mutations
Alagille syndome JAG1 or Notch1 mutations;
microdeletion or
rearrangement at 20p12
resulting in absent JAG1 gene
Noonan syndrome Mutations in PTPN11, SOS1,
RAF1, KRAS, BRAF , MEK1,
MEK2 , and HRAS
CHARGE association Mutations in CHD7 and
SEMA3E ; microdeletion at
22q11.2
Char syndrome Mutations in TF AP2B
Ellis-van Creveld
syndromeMutations in EVC or EVC2
Cardiofaciocutaneous
syndromeMutations in KRAS, BRAK,
MEK1 , or MEK2 ;
Microdeletion at
12q21.2-q22
Costello syndrome Mutations in HRAS (overlap
with Noonan and
Cardiofaciocutaneous
syndrome)
Marfan syndrome Mutations in Fibrillin-1
From [ 77]. It is open access chapter . This is an open
access chapter distributed under the terms of the Creative
Commons Attribution License ( http://creativecommons.
org/licenses/by/3.0 ), which permits unrestricted use, dis –
tribution, and reproduction in any medium, provided the
original work is properly cited. Attribution 3.0 Unported
(CC BY 3.0)Table 13.6 Non-syndromic CHD resulting from single
gene defects [ 78]
Cardiac anomalies Gene
ASD atrioventricular conduction
delay, TOF tricuspid valve
abnormalitiesNKX2.5
ASD, VSD GATA4
ASD, hypertrophic cardiomyopathy MYH6
Cardiac septation defects associated
with PHTNBMPR2
Endocardial cushion defects CREDLD1,
ALK2
BA V , early valve calcification NOTCH1
d-TGA PROSIT-240
It is an open access
Abbreviations: ASD atrial septal defect, TOF tetralogy of
Fallot, VSD ventricular septal defect, TGA transposition
of the great arteries, BAV bicuspid aortic valve, PHTN pul-
monary hypertension
M. Jinga et al.
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339t5.1
t5.2
t5.3
t5.4
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t5.6
t5.7
t5.8
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t5.10
t5.11
t5.12
t5.13
t5.14
t5.15
t5.16
t5.17
t5.18
t5.19
t5.20
t5.21
t5.22
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t5.26
t5.27
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t5.31
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t5.33
t5.34
t5.35
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t5.40
t5.41
t5.42
t5.43t6.1
t6.2
t6.3
t6.4
t6.5
t6.6
t6.7
t6.8
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t6.12
t6.13
t6.14
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t6.16
t6.17
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t6.19

protein- truncating variants (PTVs) are present
markedly in the case of patients with NS-CHD
representing one significant landmark that we
are trying to elucidate in differentiation between
NS-CHD and S-CHD [ 12].
Most gene mutations responsible for NS-CHD
and S-CHD occur in both forms (ABCC9,
ACTC1, COL1A1, NOTCH1, and NOTCH2,)
and therefore it is considered that the genetic
mechanisms of differentiation between these
forms are not known [ 79].
To sum up, these studies suggest that there
may be other gene mutations which can do
the differentiation between NS-CHD and
S-CHD. Recently genes like CDK13, CHD4, and
PRKD1 have been isolated, being involved in the
generation of S-CHD.
Conclusions
According to current evidence the causes of
CHD are exogenous and endogenous factors.
The genetic mechanisms of CHD are multiple
and include, among others, destructuring and
numerical multiplication of chromosomes,
sub- chromosomal changes (CNV), abnormal
interrelationship between transcription fac –
tors, gene destructuring, deletion and duplica-
tion, mutations, and so on. There are still
many congenital heart diseases with unknowb
etiology (61.7%). Syndrome CHDs are con –
genital malformations that include other
genetic defects while non-syndromic CHD are
clinical forms that manifest only by cardiac
involvement. Monogene or polygenes muta –
tions can produce both syndromic and non-
syndromic phenotypes and further means that
the interrelation between the original genetic
factors is not yet elucidated.
The transcription factors responsible for
cardiogenesis that regulate the differentiation
of cardiac structures are T-Box protein 5
(TBX5), GATA-binding protein 4 (GATA4)
and NK2 Homeobox 5 (NKX2.5) and the
abnormal interrelation between them gener –
ates multiple forms CHD. In the genetic
sequence of CHD the beginning damage that
has many starting points, ends with the pheno –
type specific to each genetic aberration.Far from being puzzling, knowledge of nor –
mal cardiac development and the mechanisms of
congenital heart diseases are essential to daily
practice, as much for the daily examination of
heart diseases as for genetic counselling before
birth or in the case of familial forms [ 4]. For that
reason, clinical geneticists and genetic counsel –
ors will have important responsibilities in patient
care, to make certain correct diagnosis and help –
ful communication of inheritance and recurrence
risks [ 3].
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Author Queries
Chapter No.: 13 0003405657
Queries Details Required Author’s Response
AU1 Please check if the affiliations are presented correctly.
AU2 Please note that the color shades given in this Tables 13.3, 13.4, and 13.6 has been
removed as there is no significance provided. Please confirm.
AU3 The decimal comma has been changed to a decimal point here. Please check, and
correct if necessary.
AU4 We have redrawn Figs. 3.1 and 3.4. This is for your information.
AU5 We have relabeled Figs. 13.2. Kindly check and provide better quality figures if any.