Associations with Single Rare Heterozygous NLRP7 Variants for Chinese Patients with Sporadic Gestational Trophoblastic Diseases

Background Gestational trophoblastic diseases (GTDs) encompass a group of clinically heterogeneous tumors of trophoblastic cell origin. In some populations and studies, single NLRP7 heterozygous mutations and some non-synonymous variants (NSVs) have been shown to confer genetic susceptibility to sporadic GTDs. Our study aims to investigate whether single rare heterozygous NSVs in NLRP7 predispose patients to GTDs in Chinese. Methods We performed Sanger sequencing of all NLRP7 exons and exon-intron boundaries in a cohort of Chinese patients with non-recurrent sporadic GTDs and normal childbearing women. Using in-silico analysis and several prediction programs, we investigated the effects of identied NSVs on the function of the protein. Then we examine the function in inammatory pathway to assessing the functional consequences of the identied rare NSVs in in-vitro studies. Results We found that the number of patients with rare single heterozygous NSVs that are predicted to have functional effects on the protein is signicantly higher in the rst cohort of 292 patients than in the 300 controls (p=0.028). Analysis of the validation cohort of 130 patients conrmed the signicant higher number of patients with rare NSVs than controls (p=0.0014). We show that most of the rare variants that are identied only in the patients are predicted to have functional effects on the protein by various algorithms and in-vitro experiments. Conclusion Our results demonstrate that Chinese patients with GTDs have a higher burden of single heterozygous NLRP7 variants which were predicted to have effects on the protein than controls. We suggest that some of these single NSVs may contribute to the genetic susceptibility for GTDs in China. using Fisher exact test. P values less than 0.05 were considered signicant. The potential pathogenic effects of the rare NSVs on the protein function were assessed by four different protein prediction software (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 les. 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 classied 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


Abstract
Background Gestational trophoblastic diseases (GTDs) encompass a group of clinically heterogeneous tumors of trophoblastic cell origin. In some populations and studies, single NLRP7 heterozygous mutations and some non-synonymous variants (NSVs) have been shown to confer genetic susceptibility to sporadic GTDs. Our study aims to investigate whether single rare heterozygous NSVs in NLRP7 predispose patients to GTDs in Chinese.
Methods We performed Sanger sequencing of all NLRP7 exons and exon-intron boundaries in a cohort of Chinese patients with non-recurrent sporadic GTDs and normal childbearing women. Using in-silico analysis and several prediction programs, we investigated the effects of identi ed NSVs on the function of the protein. Then we examine the function in in ammatory pathway to assessing the functional consequences of the identi ed rare NSVs in in-vitro studies.
Results We found that the number of patients with rare single heterozygous NSVs that are predicted to have functional effects on the protein is signi cantly higher in the rst cohort of 292 patients than in the 300 controls (p=0.028). Analysis of the validation cohort of 130 patients con rmed the signi cant higher number of patients with rare NSVs than controls (p=0.0014). We show that most of the rare variants that are identi ed only in the patients are predicted to have functional effects on the protein by various algorithms and in-vitro experiments.
Conclusion Our results demonstrate that Chinese patients with GTDs have a higher burden of single heterozygous NLRP7 variants which were predicted to have effects on the protein than controls. We suggest that some of these single NSVs may contribute to the genetic susceptibility for GTDs in China.

Background
Gestational trophoblastic diseases (GTDs) are de ned by the abnormal proliferation of trophoblastic cells, including hydatidiform mole (HM), invasive mole, choriocarcinoma, placental site trophoblastic tumor (PSTT) and some other rare tumors. Gestational trophoblastic neoplasia (GTNs) has been applied collectively to the latter four conditions, that have potential for invasion and metastasis and require chemotherapy and/or additional surgical procedures. GTNs may manifest in the form of high HCG levels several weeks or months after molar evacuation or as tumors demonstrated by histopathological evaluation in the form of invasive mole (IM), choriocarcinoma (CC), PSTT, and epithelioid trophoblastic tumor (ETT) [1]. GTNs occur mostly after HM, and rarely after other types of pregnancies. HM occurs once in every 600 to 1500 pregnancies in western countries, but at higher frequencies in several underdeveloped and developing countries [2][3][4]. In China, the reported incidence of HMs varies from 1 to 8.83 in every 1000 pregnancies with the highest incidence being in the province of Zhejiang where it reaches 1 in every 700 pregnancies [3,5] .
However, these data were not replicated in other populations and studies [21][22][23]. Similar studies on patients with GTNs have been limited due to their lower frequencies in western societies. To date, only one study looked at the presence of NLRP7 NSVs in patients with post-molar choriocarcinomas in Senegalese patients and showed the presence of three novel NSVs that were not found in controls from the general Senegalese population [14] .Therefore additional studies are needed to reach a clear conclusion.
NLRP7 belongs to the CATERPILLER family and contains four conserved and functional domains, including the N-terminal pyridine domain PYRIN, the Cterminal leucine rich repeat, 9 to 10 leucine-rich repeats (LRRs),the NACHT-related domain (NAD) (physical medium of oligomeric assembly) and the NACHT region in the middle of the protein (this domain contains the Walker A / Ploop motif, which is the binding site for ATP) [24,25]. The LRR and PYRIN domains on NLRP7 are involved in protein-protein interactions, and the NACHT domain is involved in apoptosis and MHC class II transactivation, and PYRIN also interacts with a variety of in ammatory components to negatively regulate in ammatory body reactions [26]. Members of the CATERPILLER protein family are widely expressed in various tissues of different species, from C. elegans, Drosophila melanogaster, rats, mice to humans [27]. They act as components of in ammation and play a role in innate immunity, in ammation and apoptosis. Patients with NLRP7 mutations have signi cantly lower levels of IL-1β and TNF-α produced by LPS stimulation and are therefore considered to function as part of the immune system [24]. Some studies suggest that NLRP7 mutations in patients can result in impaired cytokine secretion and subsequent in ammatory responses, allowing them to tolerate the growth of abnormal hydatidiform moles without embryos, thereby demonstrating the phenotype of the mole [28]. In ammasome regulate the innate immune response by promoting the maturation of in ammatory cytokines, interleukins IL-1β and IL-18. There is evidence that NLRP7 protein is also a component of In ammasome, and several proteins which is interacted with NLRP7 have been identi ed, including PRO-CASPASE-1, PRO-IL-1β, apoptosis-associated spot-like protein (ASC). CASPASE-1 mediates proteolytic cleavage of PRO-IL-1β and PRO-IL-18, affecting the biological activity, secretory form of these cytokines, to initiate and extend in ammatory host responses. CASPASE-1 recruits activated PRRs via a central in ammatory adapter, ASC, which contains the CASPASE recruitment domain (CARD) [24]. Subsequently, CASPASE-1 clustering caused its automatic activation by inducing proximity. ASC is critical for all in ammasomes that are activated by members of the NLR family that contain the PYRIN domain (NLRP) and LRR.
To address whether single heterozygous NLRP7 NSVs predispose the patients to GTDs, we sequenced NLRP7 in a rst cohort of 292 Chinese patients with GTDs (155 with GTNs and 137 without GTNs), and 300 controls. As opposed to previous studies, in which controls were screened for speci c NSVs found in patients [13,18,19,21], we sequenced the 11 NLRP7 exons in all patients and controls and found that the number of subjects with rare single heterozygous NSVs is higher in patients than in controls. We then used a validation cohort of 130 Chinese patients with GTDs (88 with GTNs and 42 without GTNs). Our data on the two independent cohorts demonstrate a higher burden of rare NSVs in NLRP7 on Chinese patients with GTDs as compared to normal bearing women. However, no signi cant differences in the number of rare NSVs were observed between patients with GTNs and without GTNs. Meanwhile, there are these 14 rare NSVs in vitro, as Glu488dup was not a single rare heterozygous NLRP7 variants, the results show that some variants have effects on the in ammatory pathway which have a positive effect predicted by various algorithms. And the variants predicted to be benign show the negative symptom.

Patients
The rst 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 classi ed 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 identi cation 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 ampli cation 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). Identi ed NSVs in patients and controls were con rmed 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 signi cant.

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 les. 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 classi ed 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 identi ed 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 con rmation by Sangon Biotech (Shanghai) .

Patients with GTDs have several rare novel NSVs
The screening of NLRP7 gene for NSVs and mutations in two independent cohorts (cohort 1, n = 292 and cohort 2, n = 130) of Chinese patients with GTDs (with GTNs and without GTNs) revealed one common NSV, V319I, with a minor allele frequency of 14.3%; three low frequency NSVs, S126,K379N and V699I, with MAF of 4.7% 1% and 1.8%, respectively; and 21 rare NSVs with MAF of < 0.5% (Table 1). All rare NSVs found in the patients were in heterozygous state with the exception of one variant in patient 525 (Supplementary Table 1). This patient was found homozygous for p.Arg721Trp, a previously reported diseasecausing mutation found in a homozygous state in three unrelated patients with RHMs, one of Armenian origin [33] and two of Indian origin (unpublished data). Another patient, 370 (Supplementary Table 1), was found heterozygous carrier of a previously reported disease-causing mutation, p.Asp722Gly, observed in a homozygous state in a Chinese patient [18]. In this study, only one patient was found to have a novel duplication of AGG leading to an in-frame insertion of a Glutamic acid at position 488 and the remaining had NSVs. This variant was not used to compute the p-values because it is not possible to predict its impact on the protein function with four software. Of the 21 rare NSVs, 14 were found only in patients and not in controls. Three of these variants are listed on the 1000 Genome database but do not have frequencies because of their scarcity and the remaining are novel. Table 1 The screening of NLRP7 gene for NSVs and mutations in two independent cohorts (cohort 1, n = 292 and cohort 2, n = 130) of Chinese patients with GTDs (wit without GTNs). And the result of NSVs predicted by four computational analysis programs, Polyphen, SIFT, AGVGD, and SNAP, that use various structural, evol comparative considerations to predict the functional impact of all identi ed NSVs.

Prediction of the pathogenic effects of the rare NSVs
To evaluate the potential functional consequences of the NSVs found in the patients and/or the controls, we used four computational analysis programs, Polyphen, SIFT, AGVGD, and SNAP, that use various structural, evolutionary, and comparative considerations to predict the functional impact of all identi ed NSVs (Table 1 and Fig. 1). The results of this analysis showed that 76% of the rare NSVs that were found only in the patients are predicted to be damaging by any of the four programs while 25% of rare NSVs observed in both patients and controls are predicted to be damaging ( Figure. 1A). This difference between the predicted pathogenicity of NSVs seen only in patients versus those seen in patients and controls was signi cant (p = 0.029). In addition, NSVs observed only in patients were predicted to have functional effects by more programs than those observed in patients and controls ( Figure. 1B). Therefore, our analysis showed that rare NSVs observed only in patients have, in general, a higher probability to be predicted damaging than those observed in both patients and controls. We also investigated the predicted impact of three variants, p.Trp66Cys, p.Cys84Tyr, and p.Met91Val, located in the pyrin domain on its recently described three-dimensional structure [28]. We found that p.Trp66Cys is highly predicted to affect the stability of the protein (Figure. 1C); the two others, p.Cys84Tyr and p.Met91Val, were also predicted to reduce the stability, but had milder effects. This was only possible for variants located in the pyrin domain for which a three-dimensional structure is available.

Patients with GTDs have a higher burden of rare variants in NLRP7
We next performed a rare variant burden test by comparing the number of subjects with rare NSVs that are predicted to have a functional effect on the protein, by any of the four programs, between patients and controls. This analysis revealed a signi cant higher number of patients with rare NSVs that are predicted to be functional than of controls (p = 0.028). We next divided the patients from cohort 1 according to the malignant degeneration of their GTDs between patients with GTNs and patients without GTNs. The analysis of mutation burden on these two categories of patients as compared to controls revealed a borderline difference between patients with GTNs and controls (p = 0.048) but no signi cance was observed between patients without GTNs and controls. Also, no signi cant difference was detected between patients with GTNs and patients without GTNs in cohort 1.
To validate the results obtained on the rst cohort, we performed the mutation burden test on a second cohort of 130 patients, 88 patients with GTNs and 42 patients without GTNs, as compared to the same 300 controls. This analysis con rmed the nding obtained on the rst cohort and demonstrated a signi cant higher number of patients with rare NSVs predicted to have functional effects on the protein than controls (p value = 0.0014). In the second cohort, a signi cant difference was detected between the number of patients with (p = 0.0256) and without GTNs (p = 0.0024) and rare NSVs predicted to be functional as compared to controls even after splitting them into the two categories. Again, in cohort 2, no signi cant difference was detected between patients with GTNs and patients without GTNs.
Some NLRP7 mutants effect the synthesis of IL-1β It has reported that, NLRP7 have an negative effect on synthesis of Pro-IL-1β, and mutation and variants in NLRP7 may disrupt this effect [13]. There are fourteen mutation variants were probably associated with GTDs, because they were found in patients and not in control subjects (Table 1). Glu488dup couldn't be built in plasmid. As shown in Fig. 2A, other thirteen mutations located in different regions of NLRP7, only one variant in PYRIN domain, ve in NACHT domain, one in NAD domain, and ve of thirteen mutants belong to LRR. To investigate the rare NSVs roles in vitro, we co-transfected the FLAG-tagged-NACHT domain, and NLRP7 mutants/wild type (we treated wild type as control), caspase-1, ASC, and IL-1β to deduce the process of the synthesis and the maturation of IL-1β. We had used western blotting to observe the difference in the protein expression. And the result indicated that patients 283, 324, respectively with T66C and V586A variants, have an augment in Pro-IL-1β (p = 0.04857, p = 0.02853), thus, there is an increased mature-IL-1β in 283 (p = 0.02116). Also, Pro-IL-1β tended to be increased in V586A, A721T, I858T, but these variants did not reach statistical signi cance (Fig. 2B).

Discussion
In these study, we identi ed a total of 21 rare NSVs only in patients which were more frequently predicted to have pathogenic effects and tended to be predicted harmful by more programs. However, no signi cant differences were detected between patients with GTNs and patients without GTNs in the two cohorts. Our results demonstrate that Chinese patients with GTDs have a higher burden of single heterozygous NLRP7 variants predicted to have effects on the protein than controls. We suggest that some of these single NSVs may contribute to the genetic susceptibility for GTDs in China.
Among the 422 analyzed patients, we found two patients with previously described pathogenic mutations, one in a heterozygous state seen as a single variant and another in a homozygous state. The latter indicates that the frequency of homozygous NLRP7 mutation among Chinese women with sporadic nonrecurrent GTDs in China is approximately 1 in 422 and is in agreement with a recent nding from England where one patient with recessive mutations in NLRP7 was found among 650 patients with a prior CHM [9].
Previous research has established an association between homozygous or compound-heterozygous maternal effect variants in the NLRP7 gene and hydatidiform moles/reproductive wastage [34]. As we know, normal NLRP7, in addition to its role in cytokine secretion, has another role in down-regulating the intracellular level of pro-IL-1β. Some mutations and variants in this gene can impair this function and consequently lead to higher amounts of pro-IL-1β production [13]. This is the pathway many studies adopted to research on RHM and reproductive loss. It is currently believed that the NLRP7 gene may cause hydatidiform moles and other types of failed pregnancies by impaired cytokine secretion and consequent in ammatory response [13]. Nevertheless, explicit mechanism has not been clari ed. So, as for GTDS, we try to investigate whether this rare NSVs of NLRP7 also have roles on this pathway. As is mentioned above, there are 13 NLRP7 mutants founded in patients and absent in control groups. And we examined them in vitro to explore any possible in uence in in ammatory pathway. The result indicated that patients 283, 324, respectively with rare variants T66C and V586A, have a higher secretion of pro-IL-1β. There was also an elevation in mature-IL-1β in patient 283. It's worth noting that, among the two variants we identify as meaningful, three bioinformatic prediction tools generated damaging results for variant T66C, which is consistent with our results above. It showed that comprehensive evaluation of pathogenicity of NLRP7 mutation through these tools has certain instructing value. As for the lack of effect on IL-1β in other 8 variants identi ed damaging by at least one of the softwares, NLRP7 may exert its pathogenicity in GTDs through other mechanisms, like affecting DNA methylation as maternal imprinted gene. Also, it is possible that our in vitro system is not sensitive enough to detect milder defects caused by these variants. There are still many features that haven't been yet elucidated, and further research is needed to reveal speci c role of NLRP7 mutations in the reproductive system.
Because of the One-Child Policy in China, our control subjects consisted of women who had only one child and no reproductive loss, which may not be a su cient criterion to exclude the potential genetic susceptibility of the control women to sporadic HMs or reproductive loss. It is, possible that some of our control women with rare NSVs may develop in the future molar pregnancies. Consequently, we believe that this limitation may have attenuated the observed differences between patients and controls. Other limitations to our study are the lack of access to another cohort of controls to validate our ndings in the controls.
Epidemiological studies have always shown that GTDs are more frequent in Asian populations [2]. The absence of differences between patients and controls in the frequencies of the common NSV, V319I, and the low frequency NSVs, V699I and K379N, suggests that these NSVs do not underlie the higher frequency of GTDs in China, which is in agreement with our previous observations on a different population [13]. On the contrary, our study suggests that rare NSVs that are predicted to be functional may underlie the genetic susceptibility for the higher incidence of GTDs in China and is in agreement with previous observations in several other diseases [35][36][37][38]. Similar analyses of large cohorts of patients with GTDs and controls from other countries and from China will be important to replicate our data and identify whether this higher burden of rare variants is speci c to some ethnic groups and populations known to have higher frequencies of GTDs than western countries.

Conclusions
Our results show that the rate of NLRP7 NSVs in Chinese GTN patients is relatively high, and the prediction of the effect of mutation on the protein is meaningful.

Consent for publication
The author con rms that the work described has not been published before and its publication has been approved by all co-authors.
Availability of data and material

Competing Interest
The authors have no con icts of interest to declare.