Subido por Rafael Gomez


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JMG Online First, published on June 7, 2017 as 10.1136/jmedgenet-2017-104611
Developmental defects
Short Report
Loss of function in ROBO1 is associated with
tetralogy of Fallot and septal defects
Paul Kruszka,1 Pranoot Tanpaiboon,2 Katherine Neas,3 Kathleen Crosby,2
Seth I Berger,1 Ariel F Martinez,1 Yonit A Addissie,1 Yupada Pongprot,4
Rekwan Sittiwangkul,4 Suchaya Silvilairat,4 Krit Makonkawkeyoon,4 Lan Yu,5
Julia Wynn,5 James T Bennett,6,7,8 Heather C Mefford,7 William T Reynolds,9
Xiaoqin Liu,9 Mathilda T M Mommersteeg,10 Wendy K Chung,5,11 Cecilia W Lo,9
Maximilian Muenke1
For numbered affiliations see
end of article.
Correspondence to
Dr Paul Kruszka, Medical
Genetics Branch, National
Human Genome Research
Institute, The National Institutes
of Health, 35 Convent Drive,
MSC 3717, Building 35, Room
1B-203, Bethesda, MD 20892,
USA; p​ aul.​[email protected]​ ih.​gov
Received 25 February 2017
Revised 6 April 2017
Accepted 19 April 2017
Background Congenital heart disease (CHD) is a
common birth defect affecting approximately 1% of
newborns. Great progress has been made in elucidating
the genetic aetiology of CHD with advances in genomic
technology, which we leveraged in recovering a new
pathway affecting heart development in humans
previously known to affect heart development in an
animal model.
Methods Four hundred and sixteen individuals from
Thailand and the USA diagnosed with CHD and/or
congenital diaphragmatic hernia were evaluated with
chromosomal microarray and whole exome sequencing.
The DECIPHER Consortium and medical literature were
searched for additional patients. Murine hearts from
ENU-induced mouse mutants and transgenic mice were
evaluated using both episcopic confocal histopathology
and troponin I stained sections.
Results Loss of function ROBO1 variants were
identified in three families; each proband had a
ventricular septal defect, and one proband had tetralogy
of Fallot. Additionally, a microdeletion in an individual
with CHD was found in the medical literature. Mouse
models showed perturbation of the Slit-Robo signalling
pathway, causing septation and outflow tract defects
and craniofacial anomalies. Two probands had variable
facial features consistent with the mouse model.
Conclusion Our findings identify Slit-Robo as a
significant pathway in human heart development and
To cite: Kruszka P,
Tanpaiboon P, Neas K, et al. J
Med Genet Published Online
First: [please include Day
Month Year]. doi:10.1136/
Congenital heart disease (CHD) is the most
common birth anomaly, and considerable progress has been made in understanding the genetic
basis of CHD in the past two decades; however,
the aetiology of most CHD remains poorly understood. Model organisms have contributed greatly
to understanding the molecular and embryonic
basis of cardiac development. One pathway that
has been well studied in animal models, but not yet
reported in humans, is the Slit-Robo pathway.1–4
The Slit-Robo signalling pathway is involved in
embryonic and heart development across species.4
Slit-Robo signalling was found to be important
in the heart tube development of Drosophila and
the zebrafish,1 2 6 neural crest migration and adhesion,3 and membranous ventricular septum formation in the mouse.4 Slits are multidomain leucinerich repeat containing proteins that are ligands for
Robo (Roundabout) receptors and are involved in
guidance cues in heart development.7 The absence
of the Robo receptor, Robo1, has been found to
be associated with heart tube assembly defects in
zebrafish and septal defects and conotruncal anomalies in the mouse.1 4 8
Here we demonstrate an association of ROBO1
(roundabout guidance receptor 1), a key component
of the Slit-Robo pathway with tetralogy of Fallot
and septal defects in humans.
Two cohorts were screened with whole exome
sequencing. In the first cohort (proband 1), 50 individuals with CHD and their parents (trios) were
consented through the International Congenital
Heart Disease protocol (NCT01952171) at the
National Institutes of Health (NIH). Physical examination and echocardiography were conducted
at Chiang Mai University, Chiang Mai, Thailand.
A second cohort (proband 2) of 366 individuals
from the USA with congenital diaphragmatic hernia
were evaluated at Columbia University Medical
Center as part of the DHREAMS study. Proband
3 was connected through the DECIPHER Consortium and was genotyped and evaluated through the
Central Regional Genetics Service, Wellington, New
Zealand. A literature search using PubMed and the
search terms ROBO1, CHD and 3p12 deletion was
completed to add more individuals to this analysis.
Chromosomal microarray
After isolating DNA from a peripheral blood sample
using standard procedures, proband 1 was evaluated for copy number variations using Illumina
HumanExome BeadChip-12v1_A; proband two
was evaluated using an Affymetrix SNP 6.0 array.
Proband 3 underwent microarray analysis using the
Agilent SurePrint G3 ISCA CGH+SNP 4×180k
(GRCh37/hg19) array platform.
Kruszka P, et al. J Med Genet 2017;0:1–5. doi:10.1136/jmedgenet-2017-104611
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Developmental defects
Whole exome sequencing was performed on the Thailand cohort
(proband 1) at the NIH Intramural Sequencing Center as previously described.9 Exome findings were confirmed with Sanger
sequencing. For the US cohort (proband 2), targeted sequencing
on an Illumina HiSeq2500 of all of the coding exons of ROBO1
was performed on 366 subjects with congenital diaphragmatic
hernia after library preparation using molecular inversion probes
(MIPS).10 Variants were aligned, and rare variants with an allele
frequency of <1% were annotated with Annovar.
Mouse model
Two previously reported mouse models with Robo1 variants
associated with CHD are described in this report.3 8 In the
first model, mice with ENU-induced mutation were isolated
in a screen as previously described.8 The molecular lesion
(c.T809T>C, NM_019413) is predicted to convert an isoleucine residue to a threonine at position 270 of the encoded
protein (p.I270T).
In the second model, transgenic Robo1−/− mice were
created using previously described procedures.3 Immunohistochemistry for cardiac troponin I (DAPI) was applied to both
Robo1+/+and Robo1−/− mice at E18.5. In situ hybridisation for
cardiac troponin I was completed on Robo1−/− mice at E18.5.3
All experimental procedures were performed in accordance
with the UK Animals (Scientific Procedures) Act 1986 and
institutional guidelines.
Case descriptions
The proband in family 1 is the second male child of unrelated parents of Thai ancestry. There is no family history of
CHD or genetic syndromes. He presented with cyanosis and
tachypnoea at 1 year of age. Referred to cardiology at 14
months old, physical examination revealed a weight of 9.08 kg
(20th centile), height of 82 cm (95th centile), head circumference of 46 cm (40th centile), pulse oximetry of 80% and an
echocardiogram showed tetralogy of Fallot. At age 3 years,
a cardiac catheterisation showed severe valvular and subvalvular pulmonic stenosis and a left superior vena cava. Surgical
correction at 3 years of age, which included ventricular septal
Table 1
defect (VSD) closure, infundibulectomy, pulmonary vavulotomy, transanular patch and closure of a secundum atrial
septal defect (ASD), resolved the patient’s clinical cyanosis. At
age 14 years, he had normal cognition and growth parameters
were: weight of 35 kg (less than fifth centile) and a height of
156 cm (15th centile). Exam revealed dolichocephaly, micrognathia, prominent nasal root, normal ear position and rotation, high arched palate and crowded teeth.
Proband 2 is an 8-year-old female of Chinese ethnicity
conceived via in vitro fertilisation. She was diagnosed with a
congenital diaphragmatic hernia and VSD immediately after
birth. On the second day of life, the patient had a congenital
diaphragmatic hernia repair with a patch. She has had normal
development with a full-scale IQ of 107 at age 5 years and no
other congenital anomalies or health problems.
Proband 3 was ascertained through the DECIPHER Consortium where we found two deletions only encompassing ROBO1;
the other deletion was only 80 kb and thought to be benign as
it was an intronic deletion. Proband 3 is a 4-month-old male of
mixed Greek and Japanese ethnicity, conceived via in vitro fertilisation. A large membranous VSD was diagnosed during newborn
period. A microarray demonstrated a 417.75 kb deletion on chromosome 3 spanning 78653579–79071345 (hg19) that encompassed ROBO1. Parental microarray analysis has identified that
the deletion is inherited from his normal mother. He had a large
membranous VSD (repaired) and has left vocal cord paralysis
with mild subglottic stenosis and mild right bronchomalacia.
He has subtle ear anomalies, with crumpled helices and uplifted
lobes. His development to date (current age 6 months) is normal.
In the medical literature, Petek et al reported a male of
Bosnian descent with a 15 Mb deletion of six genes (PROK2,
GPR27, RYBP, PPP4R2, ROBO1 and GBE1).11 Since publication
of this study, eight additional genes have been found to be in this
interval (EIF4E3, SHQ1, GXYLT2, EBLN2, PDZRN, CNTN3,
ROBO2 and IGSF4D3).12
This individual had a secundum ASD, developmental delay
and multiple other congenital anomalies (table 1).
Sequencing results
In 50 trios with CHD from Thailand and 366 trios from the
USA, two were found to have a pathogenic variant in ROBO1
Genotype and phenotype of probands with ROBO1 loss of function variants
Patient characteristics
Proband 1
Proband 2
Proband 3
Petek et al11
ROBO1 variant
c.355C>T, p.(R119*)
c.928C>T, p.(R310*)
deletion; 417.75 kb
15 Mb deletion between marker D3S3551 and the
centromere; 46,XY,del(3)(p13p11)
De novo
De novo
Maternally inherited
De novo
Allele frequency in ExAC
CADD score
Cardiac phenotype
Current age
14 years
8 years
4 months
Cognitive impairment
Non-cardiac phenotype
Dolichocephaly, micrognathia, Congenital diaphramatic
prominent nasal root, high
arch palate, crowding teeth
Left vocal cord paresis, minimal Cleft lip, downslanting palpebral fissures, square
facial dysmorphism
fancies with wide forehead, hypoplastic supraorbital
ridges, arched eyebrows, short nose, low-set
posteriorly angulated ears, short neck, narrow
shoulders, mild pectus carinatum,mild rhizomelia
and hypospadias
ASDII, secundum atrial septal defect; CADD, combined annotation dependent depletion; ExAC, exome aggregation consortium; ToF, tetralogy of Fallot; VSD, ventricular septal
Kruszka P, et al. J Med Genet 2017;0:1–5. doi:10.1136/jmedgenet-2017-104611
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Developmental defects
(proband 1 and proband 2). Proband 1 has a de novo variant
in ROBO1, c.355C>T, p.(R119*) (NM_002941.3), identified
using whole exome sequencing. An additional de novo missense
variant was found in KLHL38 (c.356T>C, p.L119P), a gene not
associated with cardiac development or human disease. There
were no rare variants in genes previously known to be associated with CHD. Proband 2 has a de novo variant in ROBO1
(c.928C>T, p.(R310*) (NM_002941.3) identified by targeted
sequencing of ROBO1. Chromosomal microarrays were normal
in both probands 1 and 2, and next-generation sequencing findings were confirmed with Sanger sequencing. In proband 3, a
418 kb deletion (Chr3:78653579–79071345; hg19) on 3p12.2
encompassing exons 4–29 (total of 31 exons; NM_002941).
Parental analysis identified this to be inherited from his unaffected mother.
Mouse model
Mice harbouring an ENU-induced mutation in Robo1 showed
both craniofacial and cardiac anomalies (figure 1). The newborn
Robo1I270T mutant exhibited shortened snout (figure 1B)
with micrognathia (figure 1B) and cleft palate (figure 1D), as
compared with that of a normal newborn mouse (figure 1A,C).
Heart malformations in the newborn Robo1I270T mutant included
double outlet right ventricle (DORV) with perimembranous
VSD (figure 1E and see online supplementary video 1), atrioventricular septal defect (figure 1F), muscular VSD (figure 1G),and
secundum ASD (figure 1H). The DORV is better visualised in
the 3D reconstruction of the heart, showing connection of both
the aorta and pulmonary artery to the right ventricle (see online
supplementary video 1). In the transgenic Robo1−/− mice
(figure 1I–L), perimembranous VSDs can be seen at embryonic
day (E)18.5 just before birth.3
We report three unrelated patients with ROBO1 loss of function variants and cardiac septation and conotruncal defects and
review a previously reported case with a 15 Mb deletion that
comprised 14 genes including ROBO1. These 14 genes have
not been previously associated with CHD, and only ROBO1
has been associated with CHD in the animal model. Interestingly, ROBO2 was included in this deletion and has been
found to be expressed in the developing mouse heart; however,
Mommersteng et al demonstrated that ROBO2 mutants lack
heart defects.3 The two patients with non-sense variants have
normal development but several other anomalies including
minor facial anomalies in proband one and a congenital
diaphragmatic hernia in proband 2. These are the first reported
patients in the medical literature with ROBO1 loss of function
variants except for large microdeletions encompassing multiple
contiguous genes.11
Although the most consistent finding among patients with
ROBO1 variants is CHD, proband 1 (table 1) and the patient
reported by Petek et al11 (table 1) have craniofacial findings consistent with the ENU-induced Robo1I270T mouse
(figure 1B,D). The finding of diaphragmatic hernias in proband
3 suggesting a role for Slit-Robo pathway in development of the
diaphragm is supported by the finding of congenital diaphragmatic hernias in mice deficient for Slit3, a ligand of Robo1.13 14
Recovery of more patients with ROBO1 variants are needed to
confirm if these patients have syndromic versus isolated CHD.
ROBO1, a transmembrane receptor, along with its Slit
ligands is part of the Slit-Robo pathway involved in cardiac
development. Morpholino (MO) knockdown of robo1 in
Kruszka P, et al. J Med Genet 2017;0:1–5. doi:10.1136/jmedgenet-2017-104611
the zebrafish is associated with inhibition of endocardial and
myocardial migration resulting in unfused heart fields in early
embryogenesis.1 Interestingly, Fish et al found that subphenotypic doses of robo1 and vegfa MOs together in the zebrafish
(low doses that cause only subtle effects when injected alone)
resulted in delayed heart field fusion, suggesting an in vivo
interaction between robo1 and vegfa.1 In both the Robo1
knockout and ENU mutant mouse, membranous ventricular
septum and overriding aorta were observed.4 All probands
in this study had septal defects, but interestingly, the mouse
model exhibited double outlet right ventricle/overriding aorta,
phenotypes that are similar to tetralogy of Fallot reported in
proband 1.3 Additionally, Robo1 was found to be expressed
in Isl1 mouse model second heart field, which is significant,
as both second heart field and neural crest defects can cause
outflow alignment problems and septal defects.4 Whereas Fish
et al reported robo1 was associated with vascular endothelial growth factor (VEGF) signalling pathway, Mommersteeg
et al showed a connection of Robo1 with the Notch signalling pathway, with loss of Robo1 associated with decreased
expression of Notch1, Notch2 and Hey1.3 Both of these pathways have been associated with CHD. VEFG is known to be
important for heart development, and VEGF variants have
been associated with tetralogy of Fallot15 and VSD.16 Notch
signalling and both NOTCH1 and NOTCH2 have been associated with ToF.17 Heart formation requires the function of
multiple pathways including cross-talk between Notch and
VEGF,18 and ROBO1 has been shown to be related to both
signalling pathways.
Like many CHD genes, there may be other factors such as
gene–gene and gene–environment factors that affect phenotype, which is seen in the variable presentation in both humans
and the two different mouse models described above. ROBO1
loss of function mutations were found in 2 of 416 trios
screened in this study compared with 25 loss of function variants found in the ExAC database (approximate 60 000 exomes
of presumably healthy individuals).19 Although this is a significant difference in the ratio of loss of function variants in our
study compared the ExAC database (p<0.05; Fisher’s exact
test), other factors such as genetic modifiers may be involved
in the clinical phenotype of CHD. Additionally, although the
ExAC cohort is presumed to be relatively healthy, the possibility exists that some of the individuals with loss of function
variants had septal defects that were repaired or that closed
spontaneously without intervention.
In conclusion, we show that variants in ROBO1 are associated with CHD involving septal defects and tetralogy of Fallot.
Author affiliations
Medical Genetics Branch, National Human Genome Research Institute, The National
Institutes of Health, Bethesda, Maryland, USA
Division of Genetics and Metabolism, Children’s National Health System,
Washington, DC, USA
Genetic Health Service New Zealand (Central Hub), Wellington, New Zealand
Division of Pediatric Cardiology, Department of Pediatrics, Chiangmai University,
Chiang Mai, Thailand
Department of Pediatrics, Columbia University Medical Center, New York, New York,
Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle,
Washington, USA
Division of Genetic Medicine, Department of Pediatrics, University of Washington,
Seattle, Washington, USA
Division of Genetic Medicine, Seattle Children's Hospital, Seattle, Washington, USA
Department of Developmental Biology, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania, USA
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
Department of Medicine, Columbia University Medical Center, New York, New York,
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Developmental defects
Figure 1 Craniofacial (A–D) and cardiac (E–H) defect phenotypes shown with episcopic confocal histopathology in Robo1I270T mutant mouse; (A and
C) normal newborn mouse; (B) newborn Robo1I270T mutant exhibited shortened snout (delineated by black line) with micrognathia (see arrowheads). (D)
Newborn Robo1I270T mutant with cleft palate; (E) double outlet right ventricle with perimembranous ventricular septal defect; (F) atrioventricular septal
defect; (G) a muscular VSD (arrowhead); and (H) secundum atrial septal defect (arrowhead). (I–L) Troponin I stained sections of transgenic murine heart at
E18.5. (I) Wildtype. Arrow shows ventricular septum; asterisk at entrance to aorta; plus sign at pulmonary vein; (J) Robo−/− with arrowhead shows ventricular
septal defect; and (K and L) Robo−/− with arrowhead shows ventricular septal defect. Ao, aorta; AVSD, atrioventricular septal defect; CP, cleft palate;
DORV, double outlet right ventricle; LA, left atrium; Pa, pulmonary artery; RA, right atrium; VSD, ventricular septal defect.
Contributors PK, PT and WKC designed the study. PT, KN, KC, YP, RW, SS, KM and
JW collected data. SIB, YAA, LY, HCM, JTB, PK and WKC performed next-generation
sequencing. AFM and LY performed Sanger sequencing. MTMM, WTR and CWL
conducted animal studies. PK, PT, WKC and MM drafted the manuscript. All authors
revised the manuscript.
Funding This work was supported by the intramural programme of the National
Human Genome Research Institute, National Institutes of Health, the Wellcome Trust
and the British Heart Foundation (PG/15/50/31594), the Burroughs Wellcome Fund
Career Award for Medical Scientists (JTB), the National Institute of Health grant
(HD057036) and by Columbia University's Clinical and Translational Science Award
(CTSA), grant (UL1 RR024156) from National Center for Advancing Translational
Sciences/National Institutes of Health (NCATS-NCRR/NIH), a grant from CHERUBS, a
grant from the National Greek Orthodox Ladies Philoptochos Society, Inc. and generous
donations from The Wheeler Foundation, Vanech Family Foundation, Larsen family,
Kruszka P, et al. J Med Genet 2017;0:1–5. doi:10.1136/jmedgenet-2017-104611
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Developmental defects
Wilke family and many other families. Funding was also provided to CWL by the
National Institutes of Health (HL098180 and HL132024). This study makes use of data
generated by the DECIPHER community. A full list of centres who contributed to the
generation of the data is available from and via email from d​ [email protected]​sanger.​ac.​uk.
Competing interests None declared.
Patient consent Obtained.
Ethics approval National Human Genome Research Institute IRB.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement We deposit phenotypic and genomic data including
medically significant findings in public databases, ClinVar or dbGAP, consistent with
NHGRI datasharing policy. Consent forms for this protocol inform participants of the
data sharing plan.
© Article author(s) (or their employer(s) unless otherwise stated in the text of the
article) 2017. All rights reserved. No commercial use is permitted unless otherwise
expressly granted.
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Loss of function in ROBO1 is associated with
tetralogy of Fallot and septal defects
Paul Kruszka, Pranoot Tanpaiboon, Katherine Neas, Kathleen Crosby,
Seth I Berger, Ariel F Martinez, Yonit A Addissie, Yupada Pongprot,
Rekwan Sittiwangkul, Suchaya Silvilairat, Krit Makonkawkeyoon, Lan Yu,
Julia Wynn, James T Bennett, Heather C Mefford, William T Reynolds,
Xiaoqin Liu, Mathilda T M Mommersteeg, Wendy K Chung, Cecilia W Lo
and Maximilian Muenke
J Med Genet published online June 7, 2017
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