Patients
The first cohort of patients contained a total of 292 unrelated Chinese patients with sporadic GTDs ascertained from various clinics and hospitals in the province of Zhejiang. We did not include any patient with RHMs in this study. Patients from cohort 1 were recruited between 2003 and 2013. The size of the initial patient cohort was based on the number of cases that were available at the time of the initiation of the study. A total of 300 controls were ascertained from the same population and ethnic group between 2006 and 2014. A second cohort of 130 patients was ascertained from the same hospitals and clinics between 2013 and 2015. Informed consents were obtained from all individuals. Questionnaires recapitulating medical and family histories was completed for every patient.
The diagnosis of sporadic HM was based on histopathological reports according to standard criteria [29]. HM can be classified into CHM and PHM. CHM were diagnosed based on the presence of abnormal circumferential trophoblastic proliferation and absence of embryonic tissues while PHMs were diagnosed based on the presence of mild trophoblastic proliferation. The diagnosis of GTNs, their staging and scoring was carried out according to the revised criteria of the International Federation of Gynecology and Obstetrics and the World Health Organization (WHO) scores [30]. These criteria are: 1) a plateau in the HCG level of four values (plus or minus 10%) recorded over a 3-week period on days 1, 7, 14, and 21; 2) an increase in HCG level of more than 10% in three tests recorded over a 2-week period on days 1, 7, and 14; 3) a persistent detectable HCG for more than 6 months after molar evacuation; 4) a histological diagnosis of choriocarcinoma or invasive mole on uterine curettage or the identification of clinical or radiographic evidence of metastases. According to these criteria, patients from cohorts 1 and 2 were divided into two groups, those with GTNs and those without GTN (Table 1). The antecedent pregnancies of patients with GTNs are provided in Supplementary Table 1, 2. Control group consisted of 300 control women of Chinese origin who had one child and no reproductive loss. All patients and controls are of Han ethnic origin.
NLRP7 mutation analysis
DNA was isolated from whole blood cells using Flexi-gene DNA Kit (Qiagen Inc. Shanghai, China).Sequence analysis was performed by PCR amplification of genomic DNA of the 11 NLRP7 exons followed by sequencing in both directions as previously described (Qian et al., 2011) at the Shanghai Biosune Biotechnology Co. Ltd (Shanghai, China). Identified NSVs in patients and controls were confirmed in a repeating PCR reaction and in available parents. Variants were annotated according to GenBank accession numbers NM_001127255.1 and Q8WX94 as recommended by the Human Genome Variation Society (http://www.hgvs.org/mutnomen/ recs.html) [31]. Protein numbering starts from the initiation codon (codon 1). We refer to NSVs for DNA changes leading to missense amino acids substitutions. We refer to common NSVs for missense variants with MAF >5%, low frequency NSVs for missense variants with 0.5%≤MAF≤5%, and rare NSVs for missense variants with MAF< 0.5% in the 300 Chinese controls.
Statistical Analysis
For each cohort of patients, statistical analyses were performed on patients with GTNs and patients without GTNs, combined or separately, as compared to the same cohort of 300 controls. The mutation burden test was performed for a single gene, NLRP7, as previously described using the collapsing method [32]. One patient (525) had a variant in a homozygous state and this patient was counted only once. Patients with more than one NSV were also counted only once and are listed in Table 1 with the least frequent variant. Chi-square analysis was performed using two-by-two contingency table at the Simple Interactive Statistical Analysis (SISA) (http://www.quantitativeskills.com/sisa/distributions/binomial.htm), using Fisher exact test. P values less than 0.05 were considered significant.
Prediction of pathogenic impacts
The potential pathogenic effects of the rare NSVs on the protein function were assessed by four different protein prediction software programs: PolyPhen-2 (Polymorphism Phenotyping v2) (http://genetics.bwh.harvard.edu/pph2/), SIFT (sorts intolerant from tolerant) (http://sift.jcvi.org/), AGVGD (Align GVGD) (http://agvgd.iarc.fr/), and SNAP. PolyPhen-2 prediction is based on sequence similarity, phylogenetic conservation and structural information. Polyphen-2 score ranges from 0 to 1 where 0 is neutral and 1 is the highest damaging. SIFT is a sequence homology based prediction tool that sorts intolerant from tolerant using multiple alignment files. SIFT score ranges from 0 to 1, where 0 is predicted not to be tolerated and 1 is predicted to be tolerated. AGVGD uses both biophysical characteristics of amino acids and multiple sequence alignment of proteins. AGVGD scores are classified in to 7 classes C0, C15, C25, C35, C45, C55 and C65, where C65 is predicted most likely damaging and C0 most likely neutral. In this study, C15 was not considered to have an effect on the protein since the AGVGD algorithm considers it as less likely to be damaging. SNAP (screening for non-acceptable polymorphisms) is a neural network based prediction tool that uses secondary structure, conservation and solvent accessibility of a protein to predict the functionality of the mutated proteins. SNAP scores range from 1 to 100 and -1 to -100. Variants with positive scores have no effects on the protein function while the ones with negative scores are functionally damaging.
Protein stability predictions were performed using four programs, I-Mutant 2.0, I-Mutant 3.0, iPTREE-STAB and Mupro. I-Mutant 2.0 and 3.0 show the free energy change (∆∆G) upon mutation. A ∆∆G of < -0.5 Kcal/mol is predicted to be largely destabilizing, ∆∆G of > 0.5 Kcal/mol is predicted to be largely stabilizing and values between the two are predicted to have a weak effect on the stability of the protein. Mupro prediction scores are shown as ∆∆G and the negative scores are predicted as destabilizing and positive as stabilizing. iPTREE-STAB uses mainly residue base information for its prediction. Again, scores are shown as ∆∆G in Kcal/mol with negative scores predicted to be destabilizing and positive stabilizing.
Experiment on functional effects of 13 rare NSVs
From two cohort of patients, there are 14 rare NSVs identified only in the patients, with one variant, Glu488dup was not a single rare heterozygous NLRP7 variants , We test the remain 13 variants of these 14 rare NSVs in vitro.
Site-directed Mutagenesis of Human NLRP7
A standard PCR was conducted with the primers in a total volume of 50μl by the following procedure: 94°C 2min, then 98 °C 10s and 68 °C 3.5min for 10 cycles, and 4 °C for one hour. Then add of DpnI (2μl, 10 U/μl) to it and incubate at 37°C for one hr. The Ki nation /ligation reactions was performed in a total volume of 20μl (DpnI-treated PCR product 2μl, PCR grade water 7μl, Ligation high 5μl, T4 Polynucleotide Kinase 1μl, at 16°C for 1hr) and then transformed the into E.coli (DH5α).All the plasmids were extracted by SanPrep Column Plasmid Mini-Prep Kit (Shanghai Sangon Biotech CO., Ltd) and the sequences were confirmation by Sangon Biotech (Shanghai) .
Cell Culture and Transfection
HEK293 cells were cultured using 12-well plates in 2ml of DMEM (Invitrogen) supplemented with 10% of FBS, when it reaches a density of 80% to 100% per well, we began the transfection. In co-transfection experiments, the pcDNA 3.1(+)-pro-IL-1β(200ng) vector encoding the human IL-1β protein, the pcDNA 3.1(+)-ASC(200ng) vector encoding the human ASC protein, the pcDNA 3.1(+)-FLAG-caspase-1(30ng) vector encoding the human caspase-1 protein (NM_033292.21) were co-transfected with pcDNA 3.1(+)-FLAG-NLRP7 (200ng). and diluted in Lipofectamine reagent according to the manufacturer’s instructions (Invitrogen). Use the indicated volume of DNA and PLUS™ Reagent with each of the four volumes of Lipofectamine® LTX.
Western blot
Incubate cells for 1–3 days at 37°C, extract Proteins from cells culture in, separated proteins with 10% SDS-PAGE, then transferred electro-phoretically onto a polyvinylidene fluoride blotting membrane. Blocked with 5% Gibco for 2 h at room temperature, and immunodetected using a monoclonal antibody directed against Flag (Cell Signaling Technology) (1:2000), and human IL-1β (2022, Cell Signaling Technology) (1:1000), and caspase-1 (2144, Cell Signaling Technology) (1:1000), and β-actin (MAB1501, Chemicon Intl.) (1:1000) overnight at 4°C. Protein bands were revealed using the Hyperfilm ECL Western blotting detection reagents (GE Healthcare) and quantified by NIH ImageJ software.