A maternal GOT1 novel variant associated with early-onset severe preeclampsia identified by whole-exome sequencing

Background This study wants to know the genetic cause of preeclampsia (PE) which is a leading cause of maternal and perinatal death, but the underlying molecular mechanisms that cause PE remain poorly understood. Many single nucleotide polymorphisms have been identified by genome-wide association studies and were found to be associated with PE; however, few studies have used whole-exome sequencing (WES) to identify PE variants. Methods Five patients with severe early-onset preeclampsia (EOPE) were recruited, and WES was performed on each patient. Sanger sequencing was used to confirm the potential causative genetic variant. Results After a stringent bioinformatics analysis, a rare variant in the GOT1 gene, c.44C > G:p.P15R, was found in one patient. Bioinformatics analysis showed that the variant site is highly conserved across several species and was predicted to be a pathogenic variant according to several online mutational function prediction software packages. Further structural biology homology modeling suggested that P15R would change the electric environment of enzymatic center, and might affect the binding affinity of substrate or product. Conclusion We demonstrated for the first time that the variant in GOT1 may be associated with EOPE, the results of this study provide researchers and clinicians with a better understanding of the molecular mechanisms that underlie maternal severe EOPE.

Here, we recruited 5 unrelated patients with severe EOPE and performed WES for each patient. Sequence variants were filtered according to standard procedures. A novel variant in GOT1 was identified in one patient (P1), and is considered to be a potentially pathogenic variant associated with PE, according to in silico analysis and structural biology predictions.

Methods
Subjects 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 [20]. 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 [21] for PE, such as those with chronic hypertension, pre-gestational diabetes, maternal body mass index (BMI) > 30 kg/m 2 , antiphospholipid syndrome/systemic lupus erythematosus (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/m 2 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 (the ratio of end-systolic and enddiastolic peak velocity of umbilical artery) 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 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/m 2 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-h 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.
Detailed clinical information for P4

WES analysis of the five patients
Five patients with severe EOPE were recruited in this study; their clinical information and data are shown in Table 1. The incidence of EOPE was about 0.5-0.8%, so the incidence of severe EOPE is much lower than EOPE. We speculated that genetic factors may play an important role in patients with severe EOPE; therefore, WES was performed for each patient. The quality of sequencing met the requirements of the bioinformatics analysis, as shown in the WES report (Supplementary Table 1).
Next, we performed a filtering process on the WES data (Supplementary Table 2). First, we retained missense, nonsense, frameshift, and splice site variants, then filtered out the variants with allele frequencies above 1% in the 1000G, ESP6500, ExAC, and gnomAD databases. We then used our inhouse database to further filter out duplicate variants, and thus obtained variants for each patient. We identified 419 SNVs and 36 insertiondeletion variants (InDels) in P1, 431 SNVs and 35 InDels in P2, 449 SNVs and 38 InDels in P3, 297 SNVs and 17 InDels in P4, and 398 SNVs and 31 InDels in P5 (Supplementary Table 2). All variants were further filtered according to the list of 40 selected candidate genes that are known to be associated with severe preeclampsia [9]. In this way, we were able to narrow down the scope of the target. In the five patients, we only identified a rare variant of the GOT1 gene c.44C > G:p.P15R in P1; the variant was confirmed by Sanger sequencing (Fig. 1a). In order to further search for genes and mutations that may be related to the occurrence of disease in other patients, we added 143 genes related to the occurrence of maternal PE to screen for mutations [9,22,23]. In total, we identified eight potentially pathogenic variants in P2, P3, and P4 that may be associated with PE (Supplementary Table 3). We found three variants in the TTN gene (Supplementary Table 3), that was consistent with the previous finding [23].

In silico analysis of the GOT1 variant
The Pro15 site is highly conserved across species from human to zebrafish (Fig. 1b), suggesting that Pro15 plays an important role in the function of the GOT1 protein.
After searching exome and genome sequencing databases, we found that the allele frequency of c.44C > G was 0 in all databases ( Table 2), indicating that the variant is extremely rare. Furthermore, several online mutational prediction tools predicted P15R to be a pathogenic variant (Table 2). We tried to use the American College of Medical Genetics and Genomics (ACMG) guidelines to classify the GOT1 variant we found. The p.P15R variant in GOT1 was classified as Uncertain Significance based on absence in large population studies (PM2), and computational predictions (PP3) according to the ACMG guidelines [24]. As the variant is heterozygous, we wanted to know whether the heterozygous variant influences disease tolerance. Constraint Metrics Z score for missense variation analysis [25] found that GOT1 was predicted to be intolerant to variation (z = 2.17, http://exac.broadinstitute.org/gene/ENSG0000012 0053). Thus, the above bioinformatics analysis suggests that the rare variant c.44C > G:p.P15R of the GOT1 gene might be potentially associated with severe EOPE.

Molecular modeling of the mutated protein
Fortunately, we found that the structure of GOT1 protein had been resolved (The Protein Data Bank, (PDB) 3ii0, Fig. 2a). By structure analysis, we found that there was an 'open pocket' at the surface of GOT1 protein, which was positive charged (Fig. 2c). The 15th residue proline was located at the edge of this pocket (Fig. 2c). In our study, this residue mutated to arginine (Fig. 2b). Arginine is a basic amino acid with a big side chain (Fig. 2d). We proposed that this point mutation would change the electric environment of enzymatic center, meanwhile, affect the binding affinity of substrate or product even more the whole process of enzyme-catalyzed reactions. Computer modeling had confirmed our proposal that P15R variant changed the electricity and enhanced the positive charge of this area (Fig. 2d).

Discussion
In this study, whole-exome sequencing was performed in 5 patients with severe EOPE. A rare variant in the GOT1 gene, c.44C > G:p.P15R was found in one patient (P1). Bioinformatics analysis showed that the variant was highly conserved across different species and was predicted to be a pathogenic variant according to several online mutational function prediction software packages. Further structural biology homology modeling suggested that P15R may change the electricity and enhance the positive charge of this area.
The GOT1 gene-full name glutamate oxaloacetate transaminase-is also known as aspartate aminotransferase. GOT1 can catalyze L-cysteine or L-aspartate substrates. Lcysteine can be converted into 3-mercaptopyruvate by GOT1, and then H 2 S can be obtained by 3mercaptopyruvate sulphurtransferase. L-aspartate can be catalyzed into L-glutamate by GOT1, which is a pyridoxal phosphate dependent enzyme found in the cytoplasm.
Previous studies suggest that maternal aspartate metabolic or catalytic processes are associated with severe preeclampsia [9]. A study using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS MRS) found that aspartate levels are higher in the placenta of patients with preeclampsia, while glutamate levels are lower [26]. Another study using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) found that glutamate levels were low in explanted placental villous fragment cultures of preeclampsia patients [27]. The P15R variant identified in the present study is located near the GOT1 enzyme activity center. Arginine is an alkaline amino acid, while both the substrate (L-aspartate) and product (L-  glutamate) involved in GOT1 reaction are acidic. The mutated protein thus, may enhance alkalinity near the active center of the enzyme, which may increase the binding ability of the substrate or the product, and in turn affect the release of the product. Thus, the decrease in L-glutamate release due to the P15R variant in the GOT1 gene could be related to the occurrence of preeclampsia in this patient.
A previous study found that hydrogen sulfide (H 2 S) is essential for a healthy placental vasculature, and that a decrease in H 2 S activity is related to the onset of preeclampsia [28]. H 2 S can cause contraction and relaxation of human umbilical vein endothelial cells [29]. The formation of hydrogen sulfide is catalyzed by GOT1, which is expressed by human umbilical artery endothelial and smooth muscle cells [29]. Thus, the P15R variant may reduce the release of 3-mercaptopyruvate, thereby affecting the production of hydrogen sulfide, which could then induce preeclampsia owing to an abnormal placental vasculature.
Previous epidemiological studies have suggested that genetic factors account for about 55-60% of PE cases, of which 30-35% can be attributed to maternal genetic factors and 20% to fetal genetic factors [11,30,31]. Therefore, one third of PE may be related to maternal genetic factors. In this study, we tried to find potential genetic pathogenic factors in 5 patients with severe EOPE, and the range of genetic genes was based on 40 gene panels related to severe EOPE. Only one patient with severe EOPE was found to have a potential pathogenic genetic variant, and no other four patients were found to have a candidate pathogenic variant. Before the study, we thought that we might find a higher proportion of genetic variants in EOPE. But at present, only 20% (one patient) of the genetic pathogenicity was found, which may be related to the small number of genes in the gene panel we used. We speculated that there may be more than 40 genes that can lead to severe EOPE. In the future, with the further study of PE genetics, the gene list  )). The whole protein is shown as cartoon in rainbow and 15th proline side chain is shown as sticks. b Close view of P15R mutant type GOT1 protein structure. The whole protein is shown as cartoon in rainbow and 15th arginine side chain is shown as sticks. c Electrostatic surface view of the wild type GOT1 protein.
Electrostatic potential is expressed as a spectrum ranging from − 64 kT/e (red) to + 64 kT/e (blue). Black arrow indicates the 15th residue proline. d Electrostatic surface view of the P15R variant in GOT1 protein. Electrostatic potential is expressed as a spectrum ranging from − 64 kT/e (red) to + 64 kT/e (blue). Black arrow indicates the 15th residue arginine will be more perfect, and more cases that we can find the cause will be found.

Conclusion
In summary, the P15R variant in the GOT1 gene identified in the present study may lead to preeclampsia by causing abnormal synthesis of glutamate or hydrogen sulfide. Furthermore, the findings suggest that abnormal aspartate metabolic or catalytic processes, together with placental vasculature contraction or relaxation abnormalities, may be associated with preeclampsia. This study provides a genetic basis for the etiology and molecular mechanisms that underlie preeclampsia.