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)