Homozygous NLRP7 mutations in a Moroccan woman with recurrent reproductive failure

To the Editor: Hydatidiform mole (HM, MIM# 231090) is an aberrant human pregnancy characterized by abnormal hyperplasic trophoblast and hydropic villi and defective foetal development (1). A rare class of highly recurrent HMs have normal diploid biparental inheritance with disrupted DNA methylation at differentially methylated regions of imprinted loci (2–5). In 2006, the NACHT, leucinerich region and pyrin domains-containing protein (NLRP) family 7 (NLRP7) gene on chromosome 19q13.42 was associated with recurrent and/or familial HMs (6). Eleven disease-associated mutations have so far been identified (7, 8). A 25-year-old woman and her 24-year-old husband were referred for genetic advice. The couple was of Moroccan origin, and there was no history of consanguinity or known pathological reproductive outcome in her family. They had experienced five reproductive failures. The first pregnancy was terminated for personal reasons at 8 weeks’ gestation and was complicated 3 weeks later by intraperitoneal haemorrhage due to trophoblastic tissue on the uterine wall. The second pregnancy was a blighted ovum spontaneously eliminated at 4 weeks’ gestation without complications. The three last pregnancies were complete HMs without embryonic structure on pathology examination. Pelvic ultrasound was normal in this patient. Biological explorations showed mild iron deficiency anaemia and presence of antinuclear antibodies (titter of 1/320, normal: .1/80) with homogeneous and speckled pattern. The remaining blood biochemistry tests (cytology, thrombophilia, immunology, factor II and V genetic polymorphisms, folates, vitaminB12, homocysteine and haemoglobin electrophoresis) showed normal results. We searched for a possible genetic origin of her recurrent reproductive failures. Cytogenetic studies showed a normal karyotype for her husband. The patient’s karyotype was 46,XX, inv(14)(q21q23 31). Heterozygotes for autosomal paracentric inversions have very little risk of abnormal offspring (9), and HMs have not been reported associated with chromosome 14 paracentric inversion. So we conducted molecular analyses of theNLRP7 gene on genomic DNA extracted from the patient’s peripheral white blood cells. No tissue was available from any of her molar pregnancies. Sequencing (Fig. 1) showed that she was homozygous for the p.Arg693Trp (c.2077C . T) mutation that was previously reported in a German family (MoGe2) with recurrent HM and no other kind of reproductive failure (6, 10). This variant is unlikely to be a polymorphism because it was not found in 360 control chromosomes (6). To introduce an alternative rapid screening of this gene, we also developed high resolution melting (HRM) analysis using the LightCycler 480 System. Briefly, 20 ng DNA from our patient either crude or mixed with 0.5 amount of wild-type DNA, positive samples [homozygous and heterozygous genotypes from the MoGe2 German family (6, 10)] and a reference (wild-type DNA) were amplified in parallel experiments using a 96-well LightCycler 480 Real-Time PCR System and the LightCycler 480 high resolution melting master kit (Roche Diagnostics, Mannheim, Germany). The HRM curves easily confirmed that our patient’s DNA and the MoGe2-positive control sample bore the same homozygous mutation (Fig. 1). NLRP7 mutations have been reported in seven families and six isolated patients with recurrent HM (11, 12). The exact role of NLRP7 in this pathology is poorly understood. In an in vitro study with the overexpression of NLRP7 and other partner proteins, the wild-type protein acts as an inhibitor of interleukin-1b, a pro-inflammatory cytokine abundantly expressed in the reproductive tract. Mutation in NLRP7 would impair the implantation and development of the embryo, by