PE can be characterized into 2 different disease entities: early-onset PE (EOPE, corresponding to preeclampsia registered at < 34 weeks) and late-onset PE (LOPE corresponding to preeclampsia registered at > 34 weeks). They are associated with different fetal and maternal effects, heritability, biochemical markers, and clinical symptoms . We chose patients diagnosed with EOPE at the Beijing Obstetrics and Gynecology Hospital between January 2018 and June 2018. The diagnostic criteria of severe PE include: blood pressure of 160/110 mmHg or higher; thrombocytopenia (platelet count below 100,000/μL); impaired liver function as indicated by abnormally elevated blood concentration of liver enzymes; severe persistent right upper abdominal pain; progressive renal insufficiency (serum creatinine concentration above 1.1 mg/dL or doubling of serum creatinine concentration); pulmonary edema; and cerebral or visual disturbances. Women with high risk factors  for PE, such as those with chronic hypertension, pre-gestational diabetes, maternal body mass index (BMI) >30 kg/m2, antiphospholipid syndrome/SLE, or who had received assisted reproduction, were excluded from our study. However, we did not rule out women with a prior history of pre-eclampsia. Using the above criteria, we identified 5 patients with severe EOPE (Table 1).
All procedures involving human participants were performed in accordance with the ethical standards of the Ethics Committee of Beijing Obstetrics and Gynecology Hospital and with the 1964 Helsinki declaration and its later amendments. Written informed consent was obtained from each patient.
Detailed clinical information for P1
Patient No. 1 (P1) is a 35-year-old woman, body weight 62.5 kg, with a BMI of 22.9 kg/m2 prior to pregnancy. Six years ago, she delivered a baby girl by cesarean section because the umbilical cord was wrapped around the fetus’s neck. During the same period, she was diagnosed with severe PE (gestational age of onset is unknown), and her blood pressure returned to normal postpartum. She refused to provide a personal medical history or a family history. The patient’s blood pressure rose to 140/90 mmHg at 25 weeks of gestation, and an ultrasound examination showed that the fetus was small. She began to experience limb edema at 26 weeks of gestation but was not treated at this time. At 28 weeks of gestation, her blood pressure rose to 197/114 mmHg and urinary protein appeared as 3+, but she made no complaints. When the patient visited our hospital at that time, her body weight was 80 kg, and an ultrasound test showed that umbilical cord blood flow was S/D 4.8 and fetal growth was restricted. Her urinary protein went up to 4+, and 24 h urine protein was 7431.2 mg. The highest level of serum creatine was 85.6 μmol/L, blood urea nitrogen was 10.56 mmol/L, uric acid was 472.9 mmol/L, with no detected anemia or thrombocytopenia. The lowest level of albumin was 22.6 g/L. As for liver damage, the highest level of alanine aminotransferase was 52.5 U/L, aspartate aminotransferase was 41.6 U/L, and lactate dehydrogenase was 332 U/L. The highest level of d-dimer was 10.87 mg/L. Echocardiography showed no abnormalities. Both antinuclear antibodies, antibody spectrum, and cardiolipin antibody were all negative. After administering drugs and inducing labor, P1 gave birth to a stillborn child. She recovered well and was released from the hospital 10 days later.
Detailed clinical information for P2
Patient No. 2 (P2) is a 27-year-old woman who was gravida 1, para 0, and her BMI was 22.1 kg/m2 prior to pregnancy. She denied personal medical history or family history. Down screening showed high risk of open neural tube malformation at 13 weeks of gestation. The patient’s presented with high blood pressure of 178/120 mmHg during regular prenatal examination at 27 weeks of gestation, with complaint about headache, dizziness and edema of lower extremity for half a month, and nausea for a week or so. The serum creatine went up to 100.9 μmol/L within the next week. Her 24-hour urine protein quantification reached 7212.5 mg/24 h. No anemia or thrombocytopenia was found. Lupus anticoagulant and anticardiolipin antibody tests were both negative. The patient received antispasmodic treatment with magnesium sulfate, and antihypertensive therapy. However, the patient developed oliguria, pleural effusion and peritoneal effusion，then she underwent cesarean section. The weight of neonatal was 885 g. Apgar score after birth was 9’-9’-9’. After the operation, the serum creatine went up to 121.2 μmol/L, with the serum urea nitrogen 11.76 mmol/L, and the uric acid 692.3 mmol/L, and the minimum level of albumin was 24.0 g/L. The patient continued to receive antihypertensive, diuretic and prophylactic anticoagulant therapy. Finally, the patient recovered well and left hospital 6 days after the operation.
Detailed clinical information for P3
Patient No. 3 (P3) is a 29-year-old woman, who was gravida 1, para 0. Her BMI was 20.0 kg/m2 prior to pregnancy. She denied personal medical history or a family history. The patient’s presented with high blood pressure of 145/100 mmHg and her urinary examination showed urinary protein (3+) during regular prenatal examination at 27 weeks of gestation. She didn’t complain about headache, dizziness or edema. Her CVT indicated high peripheral resistance. Her 24-hour urine protein quantification could reach 3882.0 mg/24 h. Lupus anticoagulant and anticardiolipin antibody tests were both negative. The patient received antispasmodic treatment with magnesium sulfate, and antihypertensive therapy. As a result of rapidly elevating blood pressure of 180/120 mmHg and upper abdominal pain, the patient underwent urgent cesarean section. The weight of neonatal was 1110 g. Apgar score after birth was 10’-10’-10’. One day after the operation, the platelet count fell down to 66*109/L，and ALT level elevated to 110.6 IU/L, AST 50.9 IU/L，LDH 375 IU/L，Crea 90.1 μmol/L，BUN 9.03 mmol/L, UA706.8 μmol/L. Her abdominal ultrasound showed no abnormality. She was therefore diagnosed with HELLP syndrome. The patient then received antihypertensive, diuretic and prophylactic anticoagulant therapy, and she recovered and left hospital 6 days after the operation.
Detailed clinical information for P4
Patient No. 4 (P4) is a 31-year-old woman, who was gravida 2, para 0. Her BMI was 26.8 kg/m2 prior to pregnancy. And she was diagnosed with preeclampsia and intrauterine fetal death occurred at 24 weeks of gestation 3 years ago. The patient’s mother had hypertension in her old age. The patient’s presented with high blood pressure of 140/90 mmHg complaining about headache and dizziness for three weeks at 26 weeks of gestation. Her 24-hour urine protein quantification could reach 1300 mg/24 h. Ultra-sound test showed fetal growth restriction. Umbilical cord blood flow showed B=0, and the patient received Induction of labor by amniocentesis and rivanol. After two days of operation, the blood pressure rose to 190/124 mmHg, and the patient complained about upper abdominal pain. Biochemical tests showed that ALT 104.4 IU/L，AST 105.9 IU/L，UA 531.7 μmol/L，Crea 93.3 μmol/L，LDH 524 IU/L，ALB 31.3 g/L，routine blood test showed that her platelet account fell down to 100*109/L，the haemoglobin level was in normal range. She was diagnosed with partial HELLP syndrome. Lupus anticoagulant was positive, and anticardiolipin antibody tests was negative. The patient received antihypertensive, diuretic and prophylactic anticoagulant therapy, and she recovered and left hospital 6 days after the operation.
Detailed clinical information for P5
Patient No. 5 (P5) is a 31-year-old woman, who was gravida 1, para 0. Her BMI was 26.8 kg/m2 prior to pregnancy. She denied personal medical history or a family history. The patient developed pruritus at 28 weeks of gestation. She presented with high blood pressure of 170/120 mmHg with edema during regular prenatal examination at 30 weeks of gestation, and biochemical examinations showed that ALT 317.2 IU/L, AST 178.3 IU/L, TBA 14.4 μmol/L, LDH 495 IU/L, Crea 92.9 μmol/L, BUN 6.84 mmol/L, UA 524.7 μmol/L, ALB18.2 g/L, without hemolysis or thrombocytopenia. Her urinary protein was 3+，the 24-hour urine protein quantification could reach 2884.8 mg/24 h. BNP level was 515.6 pg/ml. And ultrasound showed that massive ascites could be found. She was diagnosed with severe preeclampsia，partial HELLP syndrome and intrahepatic cholestasis of pregnancy. Then she received emergency cesarean section at 31 weeks of gestation, and the weight of the newborn baby is 1355 g. Apgar score after birth was 10’-10’-10’. After liver protecting and antihypertensive therapies, she recovered and left hospital 5 days after the operation.
Whole-exome sequencing analysis
Genomic DNA was extracted from peripheral blood using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). Whole-exome sequencing (WES) was performed by Annoroad Gene Technology Co., Ltd, Beijing. Briefly, exomes were captured using a SureSelect Human All Exon V6 Kit (Agilent Technologies), and were sequenced using a HiSeq X10 Sequencer (Illumina). Raw Reads were mapped against the human reference genome hg19 using Burrows-Wheeler Aligner (BWA). Single nucleotide variants (SNV) were identified by SAMTools and Genome Analysis Toolkit (GATK) software, and ANNOVAR was used for SNV functional annotation and filtering. Variants fulfilling the following criteria were retained: (i) missense, nonsense, frame-shift, or splice site variants; (ii) absent from the Exome Aggregation Consortium database (ExAC, http://exac.broadinstitute.org/), Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/), 1000 Genomes (http://browser.1000genomes.org/index.html), ESP6500 (http://evs.gs.washington.edu/EVS/), and our inhouse database.
Sanger sequencing validation
The whole-exome sequencing results were validated using Sanger sequencing. For the GOT1 (c.44C>G:p.P15R) variant, forward (5′-ATTGGTTAATCGCGTTGCCAA-3′) and reverse (5′-CCACACCTGCATCTGTAAAATGG-3′) primers were used for PCR amplification and Sanger sequencing. DNA products were electrophoresed on an ABI 3730 XL DNA sequencer (Applied Biosystems, Bedford, MA).