Molecular Genetic Analyses And Pathogenicity Of Gene NLRP7and KHDC3L Of Hydatidiform Moles

BackgroundHydatidiform mole （HM） is a benign form of gestational trophoblastic diseases characterized by swelling chorionic villi and abnormal proliferation of trophoblasts. According to histopathological features, HMs can be classified into two types:complete hydatidiform moles （CHMs） and partial hydatidiform moles （PHM）. CHM are commonly androgenetic diploid （androgenetic CHM, AnCHM）, while lately several cases of biparental CHMs （BiCHM） have been reported. BiCHM is often found with familial recurrent hydatidiform mole （FRHM）, which is rare and considered to be autosomal recessive inherited. Patients with FRHM commonly have abnormal CpG methylation in imprinted genes, and several homozygous mutations have been detected in candidate pathogenic gene NLRP7and KHDC3L of such patients.ObjectivesTo determine the genetic origin of FRHM, RHM and sporadic HM by short tandem repeat （STR） polymorphism detection of HMs and their parents. To confirm the association between FRHM and BiCHM. To screen for mutations or variants of coding sequence of NLRP7and KHDC3L in BiCHM patients using normal female relatives and AnCHM patients as control. To investigate the pathogenic potential of suspected mutations or variants by searching in corresponding databases.MaterialsClinical information of1pedigree of FRHM,5pedigrees of RHM and6sporadic patients with HMs, as well as fresh molar tissues or formalin-fixed paraffin-embedded blocks of molar tissue and blood samples of patients, her husband and mother and sisters （for2pedigrees）. Extract DNA from collected molar tissues and blood.Methods1. Confirm diagnoses of hydatidiform moles by reviewing hematoxylin-eosin （HE） staining and immunohistochemical staining of p57KIP2of each specimen, and differentiate CHM from PHM.2. Perform PCR amplification of HM’s and their parents’ DNA, using fluorescence- labeled standard primers of STRs located on different chromosomes. Detect STR polymorphism of PCR products by polyacrylamide gel electrophoresis and DNA sequencer, and analyze by groups of HMs and their parents to determine the genetic origin of HMs.3. Perform PCR amplification of HM patients’DNA （and their mother’s and sister’ if available） using primers of coding sequences of NLRP7and KHDC3L. Sequence the PCR products and compare them with reference sequence （NM₂06828.3and NM₀01017361.2） to look for mutations or non-synonymous variants （NSVs）. Search SNP database to check if the mutations or NSVs were novel.Results15hydatidiform mole specimens were collected, among which1was FRHM,8were RHM and6were sporadic HM.1FRHM and1RHM without other normal conception were BiCHMs;5RHMs and6sporadic HMs were AnCHM, among which8were homozygote and3were heterozygous;2RHMs were PHMs.3NSVs （c.955G>A, c.1280T>C and c.1441G>A） were found in coding sequence of NLRP7and1NSV （c.602C>G） in KHDC3L after screening15cases with RHMs or sporadic HM. c.1441G>A of NLRP7only existed in one BiCHM patient. Two cases with both c.955G>A and c.1280T>C of NLRP7have recurrent AnCHM or spontaneous abortion （SA）. Homozygous c.602C>G of KHDC3L was found in HM patients and a healthy woman with no HM or SA.ConclusionFRHM is BiCHM, and RHM without normal conception can be BiCHM. RHMs with normal conception and sporadic HMs are AnCHM or PHM. BiCHM has indistinguishable clinical and histopathological manifestations compared with AnCHM, while molecular genetic analysis can differentiate them.NSV c.1441G>A of NLRP7might contribute to the onset of BiCHM. However, there is no clear pathogenic mutation or NSV in coding sequence of NLRP7and KHDC3L of HM patients in this study. BiCHM has genetic heterogeneity, and there might be other pathogenic genes besides NLRP7and KHDC3L.The method of molecular genetic analysis and gene sequencing used in this study can be introduced to clinical practice to determine the genetic origin of HM and provide NLRP7and KHDC3L DNA testing for patients with at least two HMs.