Gene Mutation Detection And Analysis In Nephronophthisis Patients

BackgroundNephronophthisis(NPHP;MIM256100) is an autosomal recessive, chronictubulointerstitial nephropathy that represents the most common genetic cause ofend-stage renal disease (ESRD) in children and adolescents. It was first described byFanconi et al.in1951. It has been estimated that NPHP has an incidence of1:50,000live births in Canada and1:1,000,000inhabitants in the United States. The disease canbe subdivided clinically based on the age of onset of ESRD into infantile, juvenile andadolescent categories with the median age of onset being1,13and19years of age,respectively. Research data from abroad showed that NPHP approximately accountedfor10%to14%in chronic kidney failure in children. And juvenile nephronophthisiswas the most common phenotype, responsible for6%to10%of all ESRD in theEuropean. There is no presice statistics about NPHP in domestic. Prior to ESRD,clinical symptoms are non-special,include polyuria, polydipsia and anemia and so on.The ultrasound findings of kidneys from NPHP patients showed generally normal orreduced kidneys size, increased echogenicity, lack of corticomedullary differentiationand later in the course of the disease cysts at the corticomedullary border of thekidneys with the exception of infantile NPHP, which showed additional featuresreminiscent of autosomal dominant or recessive polycystic kidney disease, such askidney enlargement.The histopathology is characterized by the triad of periglomerularand interstitiall fibrosis, tubular basement membrane disintegration and dilation of tubules with cyst formation mainly at the corticomedullary border.Due to the hidden onset of the disease, and clinical mainfestations,which onlyshowed symptoms of chronic renal failure, without obvious hematuria and proteinuriaor hypertension, were non-special.It belongs to the category of cystic renal disease,but cysts often appear at the later period,even not detected by imageologicalexamination. If there exists typical clinical manifestations, and renal biospy showesperiglomerular and interstitial fibrosis, tubular basement membrane thickening,thinning or fracture, tubular epithelial cells atrophy and tubular cystic expansion andglomerular sclerosis and hyaline degeneration, can be diagnosed NPHP. However, thepathological change is not always typical. So some scholars have put forward thatrenal pathology was non-special, only assisting diagonsis.In recent years, with therapid development of molecular biology research, genetic diagnostics for NPHP hasbeen projected extensively and deeply abroad, so scholars believe that moleculargenetic testing is an important method for definite diagnosis of NPHP and avoidsinvasive diagnostic measures like renal biopsy. Meanwhile, molecular genetic testingis beneficial for the prenatal diagnosis, genetic consultation and eugenics. And theindentification of more and more caustive genes is help for revealing the pathogenesisof the disease, which will lay the foundation of gene targeted treatment in future.ObjectiveThe aim of this research is to examine genes and analyze the mutations in6presumed diagnosis of NPHP patients, provids the basis for clinical diagnosis andoffers genetic guidance for the next births.MethodsThe six unrelated children who were clinical diagnosis of nephronophthisis alongwith their seven relatives,were studied. Genomic DNA was extracted from peripheralwhite blood cells. At first, multiple microsatellite markers and interal control markersin NPHP1gene were amplified by polymerse chain reaction(PCR). Whetherhomozygous deletions existing or not and deletions in size depended on amplifiedproducts of each marker. If not, amplifying all coding exons and exon-intronboundaries of the NPHP1, NPHP4, NPHP5(IQCB1), NPHP2(INVS) and NPHP3 genes in order. And all PCR products were sequenced directly by forward and reverseprimers. Ten adults with normal urine results were studied as control.ResultsThe clinical features of patients: All the five patients were male, except a female,whose ages at diagnosis range from7months to13years. Four patients are consistentwith diagnosis of juvenile nephronophthisis, and two fulfill the infantilenephronophthisis. Only one patient’s uncle affected renal cystic disease, which wasnot clear in detail, and has carried out kidney transplantation for ESRD. Six patientswere growth retardation. Two of them had a obvious history of polydipsia, polyuria.Two visited the doctor with pale complexion and fatigue. One was in hospital forrenal abormality. One was found growth retardation. One patient was made diagnosisof amblyopia at preschool period,and another one showed maculopathy by fundusexamination. The remaining were normal by fundoscopy. In5patients,24hours urineprotein quantitation was0.57±0.69g. Two patients were in renal insufficiency phaseIII, one in renal insufficiency phase IV, and three were in renal failure. The renalultrasound showed that reduced kidneys in five of them, except one normal kidneysize, increased echogenicity in cortex, obscure corticomedullary differentiation andfour patients were found multiple small cysts in kidneys. Enhanced ComputerizedTomography scan in three patients displayed small kidneys size and multiplemicrocystic lower density shadow. Three were performed Magnetic ResonanceImaging, which showed kidneys reduced in size and there were multiple scattertingpoint abnormal signal in renal parenchyma, almost in cortex.The results of genes detections: All the six patients arose PCR products of themultiple microsatellite and internal control markers in NPHP1gene, which showedthere were not NPHP1large homozygous deletions. In four juvenile nephronphthisispatients, we did not detect point mutations in all exons of NPHP1and NPHP3genes.Three of them were found heterzygous mutation of NPHP4gene IVS20-2A>T, inwhom one was complex heterzygous mutations of NPHP4geneIVS2-4delC/IVS20-2A>T and complex heterzygous mutations of NPHP5genec.-147_-141delTGGGAG/c.1301G>A. In two infantile nephronophthisis, we did not detect any mutation in all exons and splicing sites of NPHP2and NPHP3genes.Conclusions1. We shoule consider patients as nephronophthisis,who shows non-specific clinicalsymptoms including polydipsia,polyuria,anemia and growth retardation, with serumcreatinine elevating by laboratory examination,and imageological examinationdisplaying cysts at the corticomedullary border of the kidneys or not.According tothe clinical classification,we can perform gene detection,which can not only avoidinvasive measure,but also help for genetic consultation and guidance.2. Four juvenile nephronophthisis were not deteceted large homozygous deteletionsand point mutations in NPHP1gene.A heterzygous mutation of NPHP4geneIVS20-2A>T may cause three patients to juvenile nephronophthisis.It is not clearwhich gene causes another one pathopoiesis.3. There isn’t mutation in all the exons of the NPHP2gene in infantilenephronophthisis.4. There must be additional locus causing infantile nephronophthisis.5. Genetic diagnosis is an important method for establishing NPHP.