Case Report, J Clin Genom Vol: 3 Issue: 5

NLRP7 mutations in Recurrent Non- Familial Molar Pregnancies: A Case Series

Rekha Aaron^*

Department of Medical Genetics, Christian Medical College, Vellore, India

*Corresponding author:
Rekha Aaron, Department of Medical Genetics, Christian Medical College, Vellore, India
E-mail: rekha.a@cmcvellore.ac.in

Received date: October 06, 2021; Accepted date: October 20, 2021; Published date: October 27, 2021

Citation: Rekha Aaron (2021) NLRP7 mutations in Recurrent Non- Familial Molar Pregnancies: A Case Series. J Clin Genom 3:5.

Abstract

Keywords: NLRP7; KHDC3L; Hydatidiform mole; mutation; recurrent pregnancy loss

Introduction

Hydatidiform mole (HM) or molar pregnancy is a gestational trophoblastic disease that arises from excessive trophoblastic proliferation. About 15- 20% of complete molar pregnancies progress into persistent gestational trophoblastic neoplasia. Although in Asia, the incidence of HM is 1 in every 387 pregnancies, in India, it is lower at 1 in 500.However, in comparison to the reported non-Asian population, both these figures are high. The risk of choriocarcinomas following a molar pregnancy is 1000 times more as compared to a normal pregnancy. Diagnosis of molar pregnancy is based on USG (Ultrasonography) and quantitative beta –human chorionic gonadotropin (ß-HCG) estimation [1]. USG reveals presence of complex intrauterine echogenic mass with tiny cystic spaces and complete lack of fetal tissue and amnion in case of complete mole . However, accurate classification of HMs is done by the immuno- histochemical analysis of p57 expression and PCR (Polymerase chain reaction)-based genotyping. Epidemiological studies have shown that genetic factors play a key role in Asians & African Americans as these predispose them to choriocarcinoma. Maternal homozygous or compound heterozygous NLRP7 pathogenic variants have been reported in association with the development of persistent trophoblastic disease and most of them are found to be diploid biparental moles[2]. The gene so named as it has an N-terminal pyrin domain (PYD), followed by a NACHT domain, a NACHT-associated domain (NAD), and a C-terminal leucine-rich repeat (LRR) region.. NLRP7 located at 19q13.4 is a member of the NLR family of proteins known to play a key role in inflammation and apoptosis. Although most often sporadic in occurrence, HM may be recurrent and familial. The occurrence of two or more complete molar pregnancies in a woman is referred to as Recurrent HM (RHM). The incidence of RHM ranges from 1.5-9.3% [3].These are biparental in origin, that is, both parents have equal genomic contribution in the formation of the mole. NLRP7 and KHDC3L, are the maternal genes that have been implicated in recurrent HM. NLRP7 was the first identified maternal effect gene implicated in RHMs and 48-80% of patients with recurrent molar pregnancies were found to have mutations in the NLRP7 gene. It has also been implicated in other forms of reproductive wastage such as spontaneous abortions, stillbirths and intrauterine growth retardation. About 10-14% of these cases have a mutation in the KHDC3L gene and very recent case reports with HM are found to have MEI1and C11orf 80 biallelic pathogenic variants.These facts emphasize the importance of DNA testing and the utility of genetic counseling in patients with RHM for choosing reproductive options including ovum donation, adoption, or surrogacy. This retrospective study was done to identify the presence of NLRP7 and KHDC3L pathogenic variants in women with recurrent pregnancy loss and previous history of recurrent molar pregnancies who had undergone genetic analysis.

Patient Details and Methods

Patients with a history of at least one molar pregnancy and miscarriage referred to the perinatal medicine clinic of Christian Medical College and Hospital, between 2014 and 2020 were included in this retrospective study. This study was approved by Institutional review board. The patient details were obtained from the electronic database, collated and analyzed. Consent was not obtained, given the retrospective nature of the study.The genomic DNA was extracted from peripheral blood samples using Qiagen Minikit method. All the 11 exons of the NLRP7 gene were amplified using specific primers designed using primer 3 software followed by sequencing in Genetic Analyser 3500. Those patients who were negative for NLRP7 variants were further sequenced for the KHDC3L gene.All identified variants were annotated and classified based on public database frequency, in silico prediction tools, and ACMG (American College of Medical Genetics & Genomics) guidelines.

Results

 There were six patients, two of them were from Bangladesh and the rest from India. Peripheral blood karyotyping was carried out in two couples.

Case 1

A 33 years old female, non-consanguineously married for eight years. Her first two pregnancies were confirmed to be molar pregnancies. Following this she had three miscarriages, requiring D&C (Dilatation and Curettage). She continued to have elevated serum beta HCG levels and was subsequently diagnosed to have gestational trophoblastic neoplasia. Peripheral blood karyotype in the patient showed balanced translocation between chromosomes 3 & 12 [46, XX,t(3:12)(p26:q13)]and the karyotype of the husband was normal. There was no family history of RHM. She underwent 4 cycles of methotrexate and folinic acid. In lieu of persistent high HCG values, she received 8 cycles of Actinomycin. The molecular analysis identified compound heterozygous missense variants in exon 6 of NLRP7 gene; [ENST00000448121] c.2077C>T (p.Arg693Trp) and another variant c.2078G>A (p.Arg693Gln) in the same exon. These variants have not been reported in gnomAD & ExAc databases and are predicted to be damaging by multiple in silico tools. Both the variants have been reported in the Human mutation gene database (CM061148 & CM094502) and reported in Clinvar as pathogenic. The variant Arg693Trp is located in Leucine rich repeat region(LRR) and proved to have a negative impact on oligomerisation potential with increase in aggresome formation. Collating all this evidence the variants were classified as likely pathogenic variants confirming the genetic diagnosis of hydatidiform mole in this patient [4].

Case 2

A 28 year old female married for 5 years came with a history of recurrent pregnancy loss. Pedigree shown in (Figure 1A). Her first pregnancy, a spontaneous conception, resulted in a miscarriage at 8 weeks. Subsequent two pregnancies were diagnosed as molar pregnancies. She had suction evacuation and received 8 cycles of chemotherapy with methotrexate and folinic acid. USG findings were suggestive of a septate uterus.Sequencing of the NLRP7 gene revealed the same missense variant c.2077C>T (p.Arg693Trp) identified in case 1 in homozygous state. Both these patients are from the same geographical location (Bangladesh).

Case 3

This was 24 years old female non-consanguineously married for 5 years, referred for evaluation of recurrent molar pregnancy. Pedigree shown in (Figure 1B). She conceived 6 months after marriage, an USG showed molar pregnancy and D&C was done. Subsequently, she conceived twice after a gap of one year. In both the pregnancies, a scan done at 8 weeks was suggestive of molar pregnancy and D&C was done. Histopathology of products of showed partial mole and complete mole respectively. She also had additional complaints of weight gain, lethargy, and intolerance to cold, menorrhagia and her TSH (Thyroid-stimulating Hormone) level was 181µIU/ml (normal:0.3-4.5 µIU/ml ) suggestive of hypothyroidism. Molecular testing of NLRP7 revealed the presence of homozygous nonsense variant c.129 G>A in exon 2 leading to premature truncation of the protein at codon 43 (p.Try43Ter). This variant lies in the pyrin domain (PYD) of the protein which is important for caspase activation & maturation of proinflammatory cytokines IL-1β and IL-18.This variant has not been reported in 1000G or ExAc databases. In GnomAD it is reported with a frequency of 0.00001(only heterozygotes). As per ACMG guidelines the variant was classified as likely pathogenic.

Cases with single molar pregnancy (n=3)

Patient 4 was a 20-year-old woman with a previous history of miscarriage and one molar pregnancy. No other comorbidities were recorded. Patient 5 was a 29-year-old female, non-consanguineous married for five years. There was no family history or other medical illness in this case. She had a history of one molar pregnancy and two previous miscarriages. Peripheral blood karyotyping revealed normal results in this couple. Patient 6 was 20yrs old female who presented with a history of 2 miscarriages and one molar pregnancy. Histopathology was reported as the gestational trophoblastic disease with complete vesicular mole. There were no pathogenic or likely pathogenic variants identified in the above three cases. On follow-up, patients 4 and 6 have had normal pregnancies with successful outcomes. Patient 1 and 5 could not be contacted for further details of obstetric outcomes. Patient 3 was planning to undergo IVF with donor ovum. Patient 2 has not tried for pregnancy yet.

Discussion

This is a case series of six patients with recurrent pregnancy loss and one or more previous molar pregnancies who underwent genetic analysis of NLRP7 and KHDC3 genes. Three patients had NLRP7 variants of which one is a founder mutation c.2077C>T (p.Arg693Trp). None of them had KHDC3L variants. The three patients detected to have NLRP7 variants also had additional findings which could have impacted their reproductive outcomes as one had a balanced chromosomal translocation, one had a septate uterus and another had hypothyroidism. However, none of these could explain recurrent HM hence genetic studies were warranted. As reported in previous studies 48-80% of patients will have mutations in NLRP7 hence patients with at least two HMs either complete or partial should be first offered NLRP7 testing followed by KHDC3L We found NLRP7 mutations in all three cases of RHM. Since first molar pregnancy may not be easily distinguished between recurrent and non-recurrent sporadic ones, molecular testing would be exceptionally helpful to determine the molar recurrence in subsequent pregnancies. So far, around 60 pathogenic variants have been reported in the NLRP7 gene. Slim et al reported a series of 10 unrelated Indian patients wherein seven patients were identified to have mutations. The authors also demonstrated a common haplotype with two mutations c.2078G>C (p.Arg693Pro)and c.2738A>G (p.Asn913Ser) revealing the presence of a founder effect in Indian patient. The authors also observed that in women harboring biallelic pathogenic NLRP7 variants, the reproductive outcome was poor whereas in men it was not so. However, reported live births from spontaneous conception in two women with NLRP7 missense mutations that cause a less severe functional impact on the protein. Recent report suggest that those with heterozygous NLRP7 mutation may also can present with a history of reproductive wastage such as spontaneous abortion however successful pregnancy is not impaired. Therefore DNA testing in RHM has major translational value related to genetic counseling and reproductive options. Studies from various groups have shown that the chances of normal live birth are very low in women with two defective alleles in NLRP7, however around 7% of patients still had normal live births. In our study, we also found that cases with one molar pregnancy who did not harbor the pathogenic variants were more likely to have successful reproductive outcomes (2/3; 67%).

In a study done by Andreasen et al, they found out that there was greater tendency of finding NLRP 7 variants in women with familial HM. In our cohort, however, none of the women had a family history of HM. The impaired NLRP7 functioning affects inflammatory response in androgenetic cells and these androgenetic cells which are complete allograft, therefore undergo uncontrolled proliferation resulting in HM. In a normal pregnancy too, chaotic mosaic aneuploidies do occur in the early embryonic stage but are checked by the normal immune response resulting in the removal of such cells or confining them to a small portion of the placenta and therefore remain unnoticed. In this series all women except one (5/6; 83%) had one or more associated miscarriages. One patient had presented only with recurrent molar pregnancies. As reported previously 10 % to 20 % had other forms of reproductive loss, mainly as spontaneous abortions. These findings suggest that some of these patients may have a genetic predisposition to such reproductive losses which may be contributed by other previously reported genes MEI1 , C11orf80, NLRP5 & PADI6 or non-favorable epigenetic changes in the oocyte genom. In patients with RHM where no genetic cause has been identified, parental contribution to molar genomes should be studied. Studies have shown that androgenetic or triploid dispermous moles have higher chances of having live births in the future in comparison to those with recurrent diploid biparental moles. This is because complete moles having diploid karyotypes which are androgenetic in origin are usually sporadic. This is, in contrast to, recurrent HM which have diploid karyotypes but are biparental in origin. Triploid dispermous moles, on the other hand, usually result in partial moles which are non-recurrent [5].

Conclusion

The present study demonstrates the importance of genetic screening in women with recurrent molar pregnancy. The likelihood of NLRP7 gene mutation is higher in cases with RHM compared to those with single molar pregnancy. The presence or absence of NLRP 7 pathogenic variants will help in further genetic counseling regarding future reproductive outcomes in these women.

Author's Contribution

RA - substantial contributions to the conception or design of the work; acquisition, analysis, interpretation & drafting of data for the work; SD- substantial contributions to the conception & revising it critically for important intellectual content & final approval of the version to be published ; MMB - conception & revising it critically for important intellectual content; GA-design of the study and manuscript revision.

Acknowledgment

We acknowledge Dr.Nisha Consultant Clinical Geneticist, Moulana Hospital, & ARMC Aegis Hospital, Kerala for referring patient 6 for genetic diagnosis.

References

1. Andreasen L, Christiansen OB, Niemann I, Bolund L, Sunde L, et al. (2013) NLRP7 or KHDC3L genes and the etiology of molar pregnancies and recurrent miscarriage. Mol Hum Reprod 19: 773-781.
2. Qian J, Deveault C, Bagga R, Xie X, Slim R, et al. (2007) Women heterozygous for NALP7/NLRP7 mutations are at risk for reproductive wastage: report of two novel mutations. Human mutation 28: 741.
3. Wang CM, Dixon PH, Decordova S, Hodges MD, Sebire NJ, et al. (2009) Identification of 13 novel NLRP7 mutations in 20 families with recurrent hydatidiform mole; missense mutations cluster in the leucine-rich region. J Med Gene 46: 569-575.
4. Cozette C, Scheffler F, Lombart M, Massardier J, Bolze PA, et al. (2020) Pregnancy after oocyte donation in a patient with NLRP7 gene mutations and recurrent molar hydatidiform pregnancies. J Assist Reprod Genet 37: 2273-2277.
5. Abdalla EM, Hayward BE, Shamseddin A, Nawar MM (2012) Recurrent hydatidiform mole: detection of two novel mutations in the NLRP7 gene in two Egyptian families. Eur J Obstet Gynecol 164: 211-215