Genetics – Heart

G1-01Fetal echocardiographic features and whole genome sequencing results of ventricular

non-compaction cardiomyopathy.

G1-02Identification of an insertion mutation in Hes1 enhancer and study on their mechanisms in

the Tetralogy of Fallot

G1-03Prenatal identification of atrioventricular septal defect and associated genomic

abnormality by fetal echocardiography and whole genome sequencing.

G1-04Whole genome sequencing for22q11 micro deletion syndrome in fetuses with congenital

heart disease.

G1-05Prenatal diagnosis of tuberous sclerosis complex using high-throughput DNA sequencing

combined with fetal echocardiography.

G1-06A novel ZIC3 gene mutation identified in patients with heterotaxy and congenital heart

disease.

G1-07Establishment of a PRKAG2 cardiac syndrome disease model and mechanism study

using human induced pluripotent stem cells.

Identification of a denovo mutation in Hes1 enhancer and study on their mechanisms in the Tetralogy of FallotYangliu Song1, shaolin Li1, Wei Sheng1, Guoying Huang1,2 *

1Children’s Hospital of Fudan University, Shanghai, China, 201102 2Shanghai Key Laboratory of Birth Defects, Shanghai, China, 201102

Abstract :PURPOSE: Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. However, the contribution of cis-regulatory mutations to TOF remains poorly understood. To address the possible involvement of regulatory variation in TOF, we searched for regulatory mutations impacting the activity of Hes1, a transcriptional repressor related to activation and transmission of NOTCH signal pathway, with well-defined roles in development of second heart field.METHODS: Using a combination of genomics and bioinformatics, we uncovered two enhancers that regulate the endogenous expression of Hes1 in heart development. We used sanger sequencing in tissue DNA of TOF patients to analyze its candidate gene sequence, and further explore the intrinsic molecular mechanism of this mutation affecting the enhancer activity.RESULTS: We identified seven patient with a heterozygous insertion mutant in an enhancer (-1505/-657bp) upstream of Hes1. Notably, we demonstrate that this single-base-pair mutation increases the activity of the enhancer to drive expression within the heart，by affecting the binding of the transcription factor retinoid X receptor alpha (RXRα) to this site. Furthermore, we found that the expression of Hes1 was significantly increased in myocardial tissue of TOF patients compared with normal human cardiac tissue.CONCLUSIONS: Our work describes the first mutation in the Hes1 enhancer and provides functional evidence that this mutation causes increased activity of this enhancer within the heart development.

Abnormal expression of Hes1 in TOF patients' myocardium: Immunohistochemistry results showed that Hes1 expression was significantly increased in TOF patients' myocardium compared with control normal human cardiac tissue.

The results of the previous study showed that there was no significant difference in the methylation status of the promoter region, so we considered that the regulatory elements may cause the increase of Hes1 expression in TOF patients. To this end, we use the website to predict the candidate enhancer and identify two candidate segments: Hes1-E1 (-289/+98) and Hes1-E2 (-267/+98).

The candidate fragments were identified as having enhancer activity: Luciferase results showed that compared with the control group (pGL3-promoter), the promoter activity was significantly increased after insertion of the candidate fragment, indicating that the fragment has enhancer activity

Found mutation on enhancer2: sanger sequencing of TOF patient tissue DNA, we found an insertion mutation (704_705 het_dupAC) on enhancer2 (-267/+98), with a frequency of 7% (n=30) . However, the mutation does not appear in normal people.

The mutation can enhance enhancer activity: Luciferase results show that the mutant enhancer activity is significantly higher than that of the wild-type enhancer.

Mutation affected the binding of RXRa to the enhancer: As shown in the figure, the co-transfection of RXRa plasmid and wild type enhancer significantly decreased the activity of luciferase, while the activities of the co-transfected RXRa plasmid and the mutant enhancer, luciferase showed no significant change or increased. The transcription factor RXRa can bind to the wild-type enhancer and inhibit its activity, while RXRa cannot bind to the mutant-type enhancer, thereby releasing the inhibitory effect and enhancing the activity of the enhancer and even increasing.

Prenatal identification of atrioventricular septal defect and associated genomic abnormality by fetal echocardiography and whole genome sequencing.

XY Hao, YE Zhang, L Sun, JC Han, & XW Liu.Department of echocardiography, Beijing Anzhen Hospital, China.

Objective:

We sought to correlate fetal echoardiographic characteristics of atrioventricular septal defect (AVSD) with genomic

abnormalities using whole genome sequencing (WGS) and whole exomal sequencing (WES). Methods: We

retrospectively reviewed 21179 fetal echocardiograms from September 2010 to June 2016 in our institution. A total of

150 fetuses were diagnosed as AVSD, in which 47 cases had termination of pregnancies by parental choices and fetal

umbilical cord and their parental peripheral blood were obtained and WGS and WES performed. Results: For the 47

fetuses with AVSD with both fetal echocardiographic and genomic data, the mean gestation age was 25±5.9 weeks

(range 16-37 weeks). In this group, 38 fetuses had complete AVSD (80.9%), 7 partial AVSD (14.9%), and 2 transitional

AVSD (4.26%). In addition, 27 fetuses (27/47, 57.4%) had associated intracardiac defects. There were 19 fetuses

(19/47,40.43%) with associated extra-cardiac anomalies: 18 in complete AVSD and 1 in partial AVSD, whereas 14

associated with both other intra-cardiac and extra-cardiac anomalies. In 47 fetuses with AVSD, DNA was extracted

successfully in 43 fetuses, but failed in 4 because of tissue degradation. Genomic studies showed positive results in 22

fetuses (51.2%), including trisomy 21 (n=11), trisomy 18 (n= 2), trisomy 13 (n=1), large chromosome duplication (n=2)

and single-gene mutation (n=6). There were 7 positive genomic studies in 27 AVSD associated with other intra-cardiac

anomalies (25.9%), 14 in 19 AVSD associated with extra-cardiac anomalies (73.7%) and 11 in 14 AVSD associated with

intra-cardiac and extra-cardiac anomalies (78.6%). Conclusion: Fetal AVSD has a high association with extra- and intra-

cardiac anomalies, especially in complete AVSD; and a high association with genomic abnormalities. Genetic testing,

which may include WGS and WES, may be considered after prenatal diagnosis of AVSD by fetal echocardiography.

Whole genome sequencing for22q11 micro deletion syndrome in fetuses with congenital heart disease.

XY Hao, XW Liu, HR Sun, YE Li, YE Zhang, YH He.Beijing Key Laboratory of Maternal-Fetal Medicine and Fetal Heart Disease, Beijing Anzhen Hospital, China.

Objective:

We sought to investigate the frequency of 22q11 microdeletion syndrome (22q11DS) in fetuses diagnosed with congenital heart disease (CHD) and to correlate 22q11DS with phenotypic CHD for prenatal parental counseling. Methods: Umbilical cord tissue specimens of 607 fetuses with CHD diagnosed by fetal echocardiography and confirmed by postmortem autopsy after the family opted for termination of pregnancy were reviewed in Beijing Anzhen Hospital from December 2012 to February 2017. Low coverage whole genome sequencing (WGS) and whole exomal sequencing (WES) were performed to ascertain 22q11DS. Results: In this cohort, 32 (5.3%) fetuses with CHD were identified with 22q11DS. Peripheral blood samples were collected from 27 couple of parents, 2 mothers and 3 fathers (8.7%) were detected with the same 22q11DS with the fetuses, and the others 22q11DS were de novo. Of the 32 fetuses with 22q11DS, 32 (12.6%) had conotruncal cardiac defect (CTD). The most common phenotypes of 22q11DS were interruption of aortic arch (type B)(IAA-B)(100%, n = 5), posterior alignment ventricular septal defect with coarctation of aorta (paVSD/CoA)(25%, n = 1), pulmonary atresia with ventricular septal defect (PA/VSD) (21.4%, n = 6), tetralogy of Fallot (TOF)(18.2%, n = 12), persistent truncus arteriosus (PTA)(17.3%, n =5), and double outlet right ventricle (DORV)(4.5%, n = 3). Conclusion: The frequency of 22q11DS is high in fetuses with CHD. Fetuses with CTD and particular phenotypes (e.g. IAA(B), paVSD/CoA, PA/VSD, etc.) had higher frequency of association with 22q11DS. The data may aid prenatal diagnosis, prognosis and parental counseling of 22q11DS in CHD.

Prenatal diagnosis of tuberous sclerosis complex using highthroughput DNA sequencing combined with fetal

echocardiographyJ. Chen, J. Wang, L. Han, X. Gu, X. hao, Z. Weng, Y. Huang, S. Ge, Y. He

Pediatrics, St. Christopher‘s Hospital for Children, Blue Bell, PA, USA; Fetal Heart Disease Maternal Fetal Medicine Research Laboratories, Beijing Anzhen

Hospital, Capital Medical University, Beijing, China;Fujian maternal and child hospital, Fuzhou,China;Tsinghua University,Peking University, Beijing, China

Tuberous sclerosis complex (TSC) may affect multiple

systems, but few prenatal symptoms appear during

pregnancy. With development of high-throughput DNA

sequencing technology, two TSC genes have been proposed

as an independent diagnostic standard. This study aimed to

determine prenatal tubercular sclerosis diagnosis and

counseling for high-risk groups of TSC using high-throughput

DNA sequencing combined with fetal heart echocardiography

(FE).

Objectives

Results

Twenty-eight families were selected. Twenty-seven families

had members and/or probands with TSC gene abnormalities,

of which five were family genetic mutations and 22 were

caused by de novo mutations. For prenatal diagnosis, TSC

gene abnormalities were detected in five of 28 fetal amniotic

fluid samples. Among these fetus, three had multiple cardiac

rhabdomyoma (CR) by FE and the remaining fetus were

negative by FE. One special family, the proband was clinically

diagnosed with TSC, but no target genes was identified in the

whole family.

Conclusions

For high-risk TSC population, target gene detection combined

with FE for prenatal diagnosis of TSC is feasible. Counseling,

prognosis and perinatal management are facilitated based on

genetic analysis and FE.

Methods

FE was performed in 28 pregnant women with a family history

of TSC or suspected familial tuberous sclerosis. Amniotic fluid

and peripheral blood samples were collected from pregnant

women or their family members. Fetuses and family members

were further analyzed for potential TSC1 and TSC2 mutations

by next-generation DNA sequencing.

3 fetus with multiple CR

TSC gene abnormalities were detected in 5

of 28 fetal amniotic fluid samples

27 of 28 families had members and/or

probands with TSC gene abnormalities

Heredity(5) 2 fetus without multiple CR

de novo(22)

Family TSC gene sequencing

Pregnant women：fetal CR found by FE, without family TSC risk factors (1)

Pregnant women with family TSC probands(24)

Pregnant women with family TSC risk factors (3)

FE

Amniocentesis/Umbilical cord blood and peripheral blood

collection

FE

G1-06

Establishment of a PRKAG2 cardiac syndrome disease model and mechanism study using

human induced pluripotent stem cellsYongkun Zhana,1, Xiaolei Sunb,1, Bin Lia, Huanhuan Caia, Chen Xua, Qianqian Lianga, Chao Lua, Ruizhe Qiana, Sifeng Chena, Lianhua Yina, Wei Shengc,

Guoying Huangc, Aijun Sunb, ,Junbo Geb, , Ning Suna,c,d,e,

Figure 1. Generation of PRKAG2-R302Q

mutant hiPSCs. A, Echocardiograms from

the elder brother ZW (upper panel)and the

younger brother ZJ (lower panel). ZW

exhibited left ventricular hypertrophy. B, The

R302Q heterozygous missense mutation was

confirmed to be present on exon 7 of the

PRKAG2 gene in the patients (ZW and ZJ),

but not in the normal control individual, by

genomic PCR and DNA sequencing. WT,

wild type. C, Representative images showing

human dermal fibroblasts (HDFs) expanded

from skin biopsies of the patients and the

healthy individual and the typical morphology

of hESC-like iPSC colony. Scale bars, 200 μm.

D, Alkaline phosphatase stainings of the HDF

derived hiPSCs. E, Immunofluorescence

analyses of pluripotency markers OCT4

(green) and SSEA4 (red) in a representative

pluripotent stem cell clones with nuclear

staining (DNA, blue). F, Quantitative real-

time PCR (QPCR) analysis of pluripotency

marker genes OCT4, SOX2 and NANOG

indicated increased expression in HDF-

derived hiPSC lines relative to their

individual parent HDFs.

Figure 2. Phenotypic characterizations of

the PRKAG2-R302Q mutant hiPSC-CMs.

A, Representative immunostaining of cardiac

troponin T (cTnT) and sarcomeric α-actinin at

day 35 after differentiation showing increased

cell size and disorganized/thickening of

myofilaments in PRKAG2 R302Q mutant

hiPSC-CMs. Scale bars, 50 μm. B,

Quantification of cell size for WT-, ZW- and

ZJ-hiPSC-CMs (n≥200, 3 lines per individual).

C, Quantification of % cells with

disorganized/thickening of sarcomeres for

WT-, ZW- and ZJ-hiPSC-CMs (n≥200, 3 lines

per individual), P < 0.05. D, Ratios of

multinucleated cells in WT-, ZW and ZJ-

hiPSC-CMs (n≥200, 3 lines per individual), P

> 0.05. E, Representative transmission

electron microscopy images of myofibrillar

organization in WT and R302Q mutant

hiPSC-CMs. Scale bars, 500 nm. Z, Z band.

Red arrows indicate the

disorganized/thickened myofibrils. Data are

shown as mean ± SEM. *P < 0.05, ***P <

0.001.

Figure 3. The gene expression and

electrophysiological phenotypes of the

PRKAG2-R302Q mutant hiPSC-CMs. A,

QPCR analyses of the expression of cardiac-

associated genes and cardiomyocyte

hypertrophic markers normalized to GAPDH

expression (n≥6, 2 lines per individual). B,

The electrical signals recorded by MEA

reflecting beating rates in WT-, ZW-, and ZJ-

hiPSC-CMs in response to epinephrine and

metoprolol treatment. C, Quantification of

beating rates followed epinephrine and

metoprolol treatment (n≥6, 2 lines per

individual). D, An extracted MEA recorded

image showing Field Potential Duration (FPD)

and Interspike Intervals (ISI). E,

Quantification of FPD in WT-, ZW-, and ZJ-

hiPSC-CMs (n≥6, 2 lines per individual). F,

Quantification of cFPD (FPD/ISI) in WT-,

ZW-, and ZJ-hiPSC-CMs (n≥6, 2 lines per

individual). Error bars represent SEM. *P <

0.05, **P < 0.01, ***P≤0.001.

Figure 4. Analyses of Ca2+ handling

properties and contractility in hiPSC-CMs.

A, Representative Ca2+ line scan graphs and

spontaneous Ca2+ transients of WT-, ZW-,

and ZJ-hiPSC-CMs at 35 days after induction

of cardiac differentiation. B-F, Quantification

of calcium handling parameters including

transient duration 50 (B), peak to peak (C),

transient amplitude (ΔF/F0) (D), time to peak

(E), and decay time (F) in WT-, ZW-, and ZJ-

hiPSC-CMs (n > 20 in hiPSC-CMs from three

lines in each group). G, Ratios of

cardiomyocytes with irregular Ca2+ transients

in WT-, ZW-, and ZJ-hiPSC-CMs (n > 20 in

hiPSC-CMs from three lines in each group).

H-J, Representative images showing the

contraction force curve measured by FelixGX

detection system in WT-, ZW-, and ZJ-

hiPSC-CMs. K, Quantification of contraction

forces in WT-, ZW-, and ZJ-hiPSC-CMs (n >

20 in hiPSC-CMs from three lines in each

group). Data are shown as mean ± SEM. *P

< 0.05, **P < 0.01and ***P < 0.001.

Figure 5. Effects of the PRKAG2-R302Q

mutation on oxidative metabolisms of

hiPSC-CMs. Mitochondrial oxidative

metabolisms of WT-, ZW-, and ZJ-hiPSC-

CMs in glucose culture, as measured by the

cellular oxygen consumption rate (OCR),

normalized to total protein. A, Real-time

respiration measurements of hiPSC-CMs and

response to injection of oligomycin, FCCP,

and finally rotenone and antimycin A. B,

Statistics of basal oxygen consumption in

WT-, ZW-, and ZJ-hiPSC-CMs (n≥6, 2 lines

per individual). C, Statistics of ATP-

dependent oxygen consumption in WT-, ZW-,

and ZJ-hiPSC-CMs (n≥6, 2 lines per

individual). D, Statistics of maximal oxygen

consumption in WT-, ZW-, and ZJ-hiPSC-

CMs (n≥6, 2 lines per individual). E, Statistics

of respiration capacity of WT-, ZW-, and ZJ-

hiPSC-CMs (n≥6, 2 lines per individual). F-I,

Quantitative PCR analyzing expression of

regulators of mitochondrial biogenesis

NRF1(F), PGC-1α(G), PPARα(H), PPARγ(I).

J, Quantitative PCR analysis of the expression

of mitochondrial transcription factor A

(TFAM). K, Quantitative PCR analysis of the

expression of fatty acid transporter CD36. *P

< 0.01, **P < .01, ***P < 0.001.Oligo,

oligomycin; FCCP, carbonyl cyanide-4-

(trifluoromethoxy) phenylhydrazone; A/R,

antimycin plus rotenone.

Figure 6. Effects of the PRKAG2-R302Q

mutation on glycogen content and AMPK

activity in hiPSC-CMs. A, PAS stainings of

glycogen deposition in WT-, ZW-, and ZJ-

hiPSC-CMs. Scale bars, 200 μm. B,

Quantification of glycogen content in WT-,

ZW-, and ZJ-hiPSC-CMs. C, Quantitative

PCR analyses of the expression of glycogen

synthase-1(GYS1), normalized to GAPDH

expression. D, Quantification of glycogen

synthase activity in WT-, ZW-, and ZJ-

hiPSC-CMs. E-F, Expression of isoforms of

glycogen phosphorylase PYGM(E) and

PYGB(F), normalized to GAPDH expression.

G, AMPK activities were determined in

hiPSCs-CMs using the AMPK activity

detection kit. H, Western blotting analyses of

the protein levels of AMPKα and phospho-

AMPKα (Thr172). I, Western blotting

analyses of the protein level of AMPK γ2 and

β-actin (internal control). Data are shown as

mean ± SEM. *P < 0.05, **P < 0.01, ***P <

0.001.

Figure 7. Influence of inhibiting AMPK

activity in hiPSC-CMs on glycogen

accumulation and cardiac hypertrophy. A,

Representative images of PAS stainings of

hiPSC-CMs without/with the treatment of 1

μM or 2μM Compound C for 7 days. Scale

bars, 200 μm. B, Expression of GYS1 in

hiPSC-CMs without/with the treatment of 1

μM or 2μM Compound C. C, Expression of

fatty acid transporters FABP3 in hiPSC-CMs

without/with the treatment of 1 μM or 2 μM

Compound C. D-E, Expression of regulators

of mitochondrial biogenesis PPARα(D),

PPARγ(E) in hiPSC-CMs without/with the

treatment of 1 μM or 2μM Compound C.

F-I, Expression of cardiomyocyte

hypertrophic markers GATA4 (F), ANF (G),

MYH7/MYH6 (H), MYL2/MYL7 (I) in

hiPSC-CMs without/with the treatment of

1mM or 2mM Compound C. Data are shown

as mean ± SEM. *P < .05,**P < .01, ***P

< .001.

AcknowledgementsThis work was supported by the National Natural Science Foundation of China (NSFC No.31571527, No. 81322003) (N.S.); the Science and Technology Commission of Shanghai Municipality (No.17XD1400300) (N.S.); the National Key R&D Program of China

2016YFC1000500, 2016YFC1305100; and the National Key Scientific Research Projects 2014CBA02003.

Abstract

Aims

PRKAG2 cardiac syndrome is a distinct form of human cardiomyopathy characterized by cardiac

hypertrophy, ventricular pre-excitation and progressive cardiac conduction disorder. However, it

remains unclear how mutations in the PRKAG2 gene give rise to such a complicated disease.

Studying PRKAG2 cardiac syndrome using disease-specific human induced pluripotent stem cell

(hiPSC) models would not only benefit the investigation of disease mechanisms, but also push

forward the progress of individualized therapeutic strategies.

MethodsTo investigate the underlying molecular mechanisms, we generated disease-specific hiPSC-derived

cardiomyocytes from two brothers both carrying a heterozygous missense mutation c.905G>A

(R302Q) in the PRKAG2 gene and further corrected the R302Q mutation with CRISPR-Cas9

mediated genome editing.

ResultsDisease-specific hiPSC-cardiomyocytes recapitulated many phenotypes of PRKAG2 cardiac

syndrome including cellular enlargement, electrophysiological irregularities and glycogen storage.

In addition, we found that the PRKAG2-R302Q mutation led to increased AMPK activities,

resulting in extensive glycogen deposition and cardiomyocyte hypertrophy. Finally we confirmed

that disrupted phenotypes of PRKAG2 cardiac syndrome caused by the specific PRKAG2-R302Q

mutation can be alleviated by small molecules inhibiting AMPK activity and be rescued with

CRISPR-Cas9 mediated genome correction.

ConclusionsOur results showed that disease-specific hiPSC-CMs and genetically-corrected hiPSC-

cardiomyocytes would be a very useful platform for understanding the pathogenesis of, and testing

autologous cell-based therapies for, PRKAG2 cardiac syndrome.

Figure 8. Reversibility of the PRKAG2

cardiac syndrome phenotypes after

correction of R302Q mutation. A, PAS

staining of hiPSCs-CMs showing reduced

glycogen accumulation after correction of

PRKAG2 R302Q mutation. Scale bars, 200

μm. B, Representative immunostaining of

cardiac troponin T (cTnT) and sarcomeric α-

actinin at day 35 after differentiation

showing decreased cell size after correction of

PRKAG2 R302Q mutation. Scale bars, 100

μm. C, Quantification of cell size for WT-

CMs, ZW-CMs, ZJ-CMs, WT-2-CMs, ZW-C-

CMs and ZJ-C-CMs (n > 110, 3 lines per

individual) 35 days after induction of cardiac

differentiation. D, AMPK activities were

determined in hiPSCs-CMs using the AMPK

activity detection kit. Data are shown as mean

± SEM. n=4 for individual cell lines *P < .05,

**P < .01, ***P < .001.