Prenatal Diagnosis And Genetic Study Of Pathogenic Genes In A Family With Congenital Nephrotic Syndrome

BackgroundCongenital nephrotic syndrome(CNS)is a very rare kind of nephrotic syndrome,which is generally defined as the nephrotic syndrome occurring within 3 months after birth,and its clinical manifestations meet the diagnostic criteria of nephrotic syndrome with large amount of proteinuria,hypoalbuminemia,severe edema and hypercholesterolemia.According to the cause of the disease,it can be divided into primary(hereditary)and secondary(non-hereditary)congenital nephrotic syndrome.Primary CNS is caused by the coding gene of the protein of glomerular filtration barrier or other related gene mutations,while secondary CNS is mostly caused by intrauterine infection or maternal disease.The CNS caused by hereditary factors occupies a dominant position.The prognosis of congenital nephrotic syndrome is very poor.Most of the cases die within half a year after birth,and some of them have already come on in utero during pregnancy.The only effective treatment is kidney transplantation.Therefore,how to carry out prenatal diagnosis and genetic counseling for the families of CNS,in order to help the families to find the pathogenic factors and then to achieve a healthy birth,is an important clinical topic.Objectives1.This study collected and analyzed the clinical datas of a congenital nephrotic syndrome family to summarize the characteristic clinical phenotypes to help the correct diagnosis and identification of CNS.2.CNS candidate pathogenic genes were screened by high throughput sequencing.3.The preliminary functional validation of the suspected pathogenic mutation sites was carried out to elucidate the genetic causes of congenital nephrotic syndrome from various perspectives.Materials and methods:1.This study collected the clinical datas of a family with congenital nephrotic syndrome,reviewed the relevant clinical examinations and laboratory results of children with CNS,and recognized the common clinical manifestations of CNS.We guide the mother in the family to strengthen prenatal examination during pregnancy,conduct serological screening and amniocentesis in the second trimester to detect neural tube defects,chromosomal structure abnormality and chromosomal copy number variations,and recognize the intrauterine clinical manifestations of the affected children and the amniotic fluid characteristics according to the results of prenatal examinations.2.Refer to the literature on hereditary CNS,we choose Beijing Kang Xu medical laboratory's targeted sequencing product which is based on high-throughput sequencing platform,and select the genetic testing kit for kidney disease(Panel)according to the present reported 270 genes associated with hereditary kidney disease to detect all exons and introns adjacent area.With the help of bioinformatics software,we select the suspicious disease-causing genes.Then,Sanger sequencing was used to isolate and verify the suspected pathogenic varation of candidate gene in the family.3.The splicing prediction website was used to compare the changes of the splicing site of NPHS1 gene before and after the varation of NPHS1 c.712+5G>A,and preliminarily predict the effect of the newly varation on the transcript.The wild-type and mutant-type plasmids of NPHS1 c.712+5G>A were constructed and transfected into 293 T cells.The total RNA of the transfected cells was extracted for RT-PCR analysis and sequencing.The transfected cell proteins were extracted and compared with the expression of nephrin protein before and after NPHS1 gene c.712+5G > A varation by Western Blot.Immunofluorescence staining was performed on Hela cells transfected with plasmids to observe the difference of nephrin protein expression before and after NPHS1 gene c.712+5G >a mutation.I-TASSER software was used to construct tertiary structure models of amino acid sequences of wild and mutant proteins,and to compare the differences between them.Results:1.The family was of Han nationality in China,with no history of inbreeding or genetic history in the family,and no clear history of infection during the two pregnancies.After birth,the first affected child in the family presented with typical nephrotic syndrome of massive proteinuria,hypoalbuminemia,edema and hypercholesterolemia.After 3 months of support treatment,the child died due to acute renal failure and severe pneumonia.Of the second fetus,maternal peripheral blood and amniotic alpha fetoprotein(AFP)were significantly higher than the same gestational level,while the specific indicator of neural tube defects,cholinesterase,was at the normal level.At the same time,fetal ultrasonography suggested that there were soft ultrasound abnormalities with slightly increased renal volume and slightly enhanced echo in the second fetus.2.Complex heterozygous variations of c.712+5G > A and c.3325 C > T of NPHS1 gene were detected in patient with CNS by high-throughput sequencing,among which c.3325C>T of NPHS1 was a major hot-spot mutation of congenital nephrotic syndrome of Finnish type(CNF),and c.712+5G > A was a new variation which has not been reported yet.Sanger sequencing was carried out among the family.The results showed that the father of the patient carried variation of NPHS1 gene c.712+5G>A,while the mother carried variation of NPHS1 gene c.3325C>T.The family had the phenomenon of genetic cosegregation,which was consistent with autosomal recessive inheritance.3.Bioinformatics analysis software found that NPHS1 gene exon 6 5?splicing receptor site may be destroyed due to NPHS1 gene c.712+5G>A varation,and it may result in error splicing of pre-mRNA.In vitro experiments it confirmed that NPHS1 gene c.712+5G>A resulted in changes in the transcriptional level of mutant-type plasmids compared with wild-type plasmids,which was specifically manifested as abnormal cDNA sequence of NPHS1 gene and a production of transcript with missing exon 5 and exon 6 sequences.Compared with wild-type plasmid,nephrin protein expression of mutant plasmid was abnormal by Western Blot.Immunofluorescence staining confirmed that compared with wild-type plasmid,nephrin protein expression of mutant plasmid was significantly reduced.The predicted results of protein tertiary structure function showed that the structure of mutant nephrin protein was significantly changed,leading to the instability of protein structure,thus losing its biological activity and biological function.Conclusion:The CNS family is in line with autosomal recessive inheritance,and the typical clinical phenotype of hereditary CNS is postnatal nephrotic syndrome with poor performance and prognosis.Abnormal elevation of AFP in maternal peripheral blood and fetal amniotic fluid during pregnancy can be used as a characteristic prenatal screening indicator for hereditary CNS.NPHS1 gene is the suspected pathogenic gene of this family.The variation of c.712+5G>A and c.3325C>T of NPHS1 gene are the suspected pathogenic varations of this CNS family.Further in vitro experiments showed that the variation of c.712+5G>A of NPHS1 gene would affect the normal splicing of NPHS1 gene,producing abnormal transcripts,resulting in abnormal expression of nephrin protein encoded by NPHS1 gene,and thereby affecting the biological activity and biological function of the protein,which is probably the pathogenic mechanism of this CNS family.