1 Data and methods
1.1 clinical features of the PA
cases
This study collected the PA family from Anhui Province, China, which was
admitted to the Department of Cardiothoracic Surgery, Shanghai
Children’s Hospital. The family had 3 generations and 2 typical
patients. The proband (III-2) was a male infant weighing 3600g borned
masure without any specific information about the disease on fetal
screening echocardioraphy. He was soon found symptoms of cyanosis of
face and mouth after birth and therefore admitted to the local hospital.
Since the patient received an anti-infective treatment after which his
symptoms were not relieved, he was referred to our hospital.
The
physical examination showed cyanosis, wheezing, and shortness of breath,
and continuous murmurs could be caught on the precordium. His
percutaneous arterial oxygen saturation (SpO2) level in the right arm,
measured using pulse oximetry, decreased to below 70% without oxygen
uptake. And the blood routine suggested WBC
20.25*10^9/L
(normal level: 8.00-12.00*10^9/L). Blood gas analysis indicated a CO2
retention and low oxygen saturation. The echocardiogram also showed a
pulmonary atresia with intact ventricular septum and patent ductus
arteriosus. He was started on prostaglandin immediately after admitted
for ductal-dependent pulmonary blood flow. Inquired about the medical
history, the proband had a history of repeated upper respiratory tract
infections and pneumonia. The proband was finally diagnosed as pulmonary
atresia with intact ventricular septum, patent ductus arteriosus and
atrial septal defect, which showed typical symptoms of this disease. He
went to the operating room for three times after which he did well with
appropriate oxygen saturations.
Further family history research revealed that the proband’s elder
brother (III- 1) had the similar symptoms (Figure 1 ). The
proband’s elder brother was diagnosed as pulmonary atresia with
ventricular septum defect based on the present history, relevant
examination as well as physical examination. And he was also cured by
operation. With whole exome sequencing, we found the proband’s mother
carries the mutant gene and had also presents related symptoms in our
hospital. She presents an obesity (BMI=26.03) , exhaustion after mild
exercises or movements. It has been reported that mutation in BMPR2
could be associated with obesity, as well as several cardiovascular
related diseases, all of which mainly present an exhaustion after
movements.
1.2 Methods
1.2.1 Diagnostic criteria of PA patients with cardiac
color
Doppler
According to the latest clinical guidelines, prenatal PA was mainly
diagnosed with four-dimensional color Doppler ultrasound, presenting an
abnormal performance. And PA could be diagnosed early in the second
phase of pregnancy (18-22 weeks) with this method. Such fetal
four-dimensional color Doppler ultrasound abnormalities mainly exist in
the pulmonary artery, tricuspid valve and right ventricle. In addition,
abnormal reflux between the various arteries of such fetuses can also be
detected. Postpartum children are assessed by measuring the size and
function of the right ventricle, the size of the tricuspid valve, and
the degree of reflux21,22.
1.2.2 Physical examination and imaging
evaluation
Physical examination and imaging assessment were performed by two
cardiothoracic surgeons as well as two imaging specialists in our
hospital. The physical examination showed cyanosis, wheezing, and
shortness of breath, and continuous murmurs could be caught on the
precordium. And the relevant auxiliary examinations before the surgery
including CT scan, electrocardiogram and X-ray of chest are presented inFigure 2 . After obtaining written consent and signature from
all participants or their legal guardians, we take venous blood from six
people in the family. The collection and use of patient biological
samples were reviewed by the Shanghai Children’s Hospital Ethics
Committee.
1.3 Whole exome sequencing and Sanger sequencing
verification
1.3.1 Extraction of genetic
DNA
Genomic DNA extraction of 2 ml peripheral blood samples were collected
from 6 family members participating according to the manufacturer’s
experimental procedure (QIAamp DNA Blood Mini Kit). Analysis of DNA
degradation and RNA contamination by agarose gel electrophoresis.
Detection of DNA purity by spectrophotometer. Then detect DNA purity by
spectrophotometer. Accurate quantification of DNA concentration. And
finally record the purity and concentration of the samples and store
them in the refrigerator at -80 °C.
1.3.2 Methods of Whole Exome Sequencing and
identification of the
variants
The SeqCap_EZ_ExomeV3 (Nimblegen) sequencing capture system was used
in the capture of the entire exome sequencing coding region. The
procedure of sequencing was done on HiSeqTM 2000, and the results were
compared with the reference to the human genome GRCh37.p5 (hg19).
Ingenuity® Variant Analysis (QIGENE) was used in variational annotation
filtering procedure. Sanger sequencing was performed on the 3500DX
Genetic Analyzer (ABI). And the results comparisons and analysis were
performed on Mutation Surveyor 4.0 (SoftGenetics) software.
1.3.3 Sanger sequencing
verification
To identify pathogenic variants, we evaluated the pathogenicity of the 6
dominant variants using bioinformatics tools, such as PCR amplification
and Sanger sequencing, and the mutation detection within the cases
including2 patients with PA and 5 other PA-free relatives. And meanwhile
verify that this mutation is co-segregating with the disease.
1.3.4 Cytogenetic analysis
After DNA extraction with the method in step 1.3.1,array
comparative genomic hybridization (aCGH) experiments were performed
using Agilent Human Genome CGH 4x180 oligonucleotide arrays (Agilent™,
Santa Clara, CA). These microarrays contain 180 000 probes with a 13kb
average probe spacing. Arrays were scanned with the Agilent scanner and
analysed with Cytogenomics 3.0.2.11 software (Agilent™). The following
databases were used for the analysis: DGV (http://dgv.tcag.ca), DECIPHER
(https://decipher.sanger.ac.uk/), OMIM (https://www.omim.org), ClinGen
(https://www.clinicalgenome.org/), Orphanet
(https://www.orpha.net/consor/cgi-bin/index.php) and PubMed (https://
www.ncbi.nlm.nih.gov)