Prenatal Genetic Diagnosis Of The Fetuses With CAKUT

Background and ObjectiveCongenital anomalies of the kidney and urinary tract(CAKUT)collectively refer to a diverse group of structural malformations that are characterized by defects in embryonic kidney development.CAKUT constitute ~20–30% of all congenital malformations and their prevalence has been estimated to range between three and six per 1,000 births.There is a spectrum of severity of CAKUT,spanning from mild hydronephrosis to unilateral renal agenesis to dysplasia.The etiology of the majority of CAKUT cases remains unknown.Advances in human and mouse genetics have contributed to increased understanding of the pathophysiology of CAKUT.Mutations in genes involved in both transcription factors and signal transduction pathways involved in renal development are associated with CAKUT.Large cohort studies suggest that copy number variants,genomic,or de novo mutations may explain up to one-third of all cases of CAKUT.One of the major challenges to the use of genetic information in the clinical setting remains the lack of strict genotype–phenotype correlation.However,identifying genetic causes of CAKUT may lead to improved diagnosis of extrarenal complications and help to evaluate renal function and prognosis of patients.Approaches to discover new genes that cause CAKUT have so far included candidate gene studies,genomewide linkage analyses,whole exome sequencing(WES),and copy number variation analyses.The last two are the most popular nowadays.Traditional techniques for the detection of large chromosomal deletions include standard karyotyping or fluorescent in situ hybridization,which can detect deletions between 500 kb and 5 Mb in length.Copy number variants(CNVs)smaller than 5Mb can be detected using high-resolution chromosomal microarray analysis(CMA),such as the array comparative genomic hybridization(array-CGH)and single nucleotide polymorphism(SNP)microarrays.However,CMA is limited to the detection of CNVs of at least 500 bp and is unable to identify balanced chromosome alterations such as point mutations and inversions.Genetic studies in unexplained diseases are generally shifting from CNV analysis to whole exome sequencing.WES attempts to cover all coding regions of the genome.The exome is estimated to comprise ~1–2% of the genome,yet contains~85% of recognized disease-causing mutations.ES is used for detecting variants in known disease-associated genes as well as for the discovery of novel gene-disease associations.In a study from the National Institutes of Health for rare and ultrarare disorders,WES provided a diagnosis in nearly 20% of cases.Here,we use standard karyotyping,CMA and WES to analyze 453 fetuses with CAKUT with/without other structural anomalies,aiming at: 1)To explore the etiologies of the CAKUT from cytogenetical,genomic and monogenic defects aspects;2)By comparative analysis of the results of the standard karyotyping,CMA and WES technology,we evaluate the advantages and disadvantages of application these technologies in CAKUT fetuses;3)Provide a theoretical basis for establishing clinical operation procedures during the genetic diagnosis and prenatal diagnosis of CAKUT.Part I Application of Standard karyotyping and CMA investigated fetuses with CAKUT Methods1、A total of 453 CAKUT fetuses and their parents were received from the Department of prenatal diagnosis in Guangzhou Women and Children’s Hospital during August 2012 through August 2016.CAKUT were divided into 11 groups: echogenic kidney(n=21),duplex kidney(n=25),MCDK(n=73),polycystic kidney disease(n=31),hydronephrosis(n=172),enlarged and echogenic kidney(n=15),dysplastic kidney(n=29),renal agenesis(n=64),ectopical kidney(n=13),horseshoe kidney(n=5)and renal cyst(n=5).According to the fetus with CAKUT with or without other abnormalities,the samples were divided into 3 groups: isolated CAKUT(n=324),CAKUT+ soft markers(n=62)and CAKUT+ other extrarenal anomalies(n=67).2、Standard karyotyping were performed on 377 CAKUT fetuses using conventional GTG-banding techniques at the 320 band level.3 、 Genomic DNA was extracted from fetuses and their parents using Salting-outing? DNA Blood Mini kits,following the manufacturers’ protocol.4、The fetuses’ DNA samples were run on Cyto Scan TM HD Array using the manufacturer’ s protocol.Array is scanned and generates intensity(CEL)file data.Then the probe level analysis on CEL file data is performed in Ch AS software(Affymetrix,California,USA).5、The CNVs detected were further aligned with known CNVs listed in database at home and databases publically available online,such as the database of DECIPHER,OMIM,DGV,UCSC,CAGdb and ISCA.6、Both of the parental DNA was detected by CMA to further confirm pathogenic CNVs and VOUS were de novo or inherited.7、Chi-square test for statistical analysis(SPSS 13.0)Results1、377 of 453 fetuses were performed on standard karyotyping.24fetuses(6.37%,24/377)were identified with abnormal karyotypes.The detection rates of isolated CAKUT,CAKUT+ soft markers and CAKUT+ other extrarenal anomalies were 3.97%(11/277),9.09%(5/55)and 17.78%(8/45),respectively.According the Chi-square test(a=0.017),the difference between the detection rates of isolated CAKUT and CAKUT+ other extrarenal anomalies was statistically significant(3.97% vs 17.78%,P=0.001).2、11 of 24 fetuses with abnormal karyotypes were detected by CMA.We found that 4 fetuses with pathogenic CNVs,1 with VOUS and 6 with benign CNVs.3、352 of 123 fetuses with normal karyotypes were detected by CMA.We found that 11 fetuses(8.94%)with pathogenic CNVs,5(4.07%)with VOUS and 6(86.99%)with benign CNVs.4、76 of 453 fetuses were detected by CMA without karyotyping.We found that 16 fetuses(21.05%)with pathogenic CNVs,3(3.95%)with VOUS and 57(75%)with benign CNVs.Among the cases with pathogenic CNVs,there were 8(10.53%)with aneuploidies,1(1.32%)with triploidy,7(9.21%)with microdeletions/ microduplications.According the Chi-square test(a=0.05),the difference between the detection rates of aneuploidies+ triploidy by CMA and standard karyotyping was not statistically significant(P=0.087).and the difference between the detection rates of microdeletions/ microduplications by CMA in fetuses without karyotyping and CMA in fetuses with normal karyotypes was not statistically significant(P=0.803).5、In 210 fetuses with CMA,the detection rates of isolated CAKUT,CAKUT+ soft markers and CAKUT+ other extrarenal anomalies were 10.88%(16/147),14.81%(4/27)and 30.56%(11/36),respectively.According the Chi-square test(a=0.017),the difference between the detection rates of isolated CAKUT and CAKUT+ other extrarenal anomalies was statistically significant(10.88% vs 30.56%,P=0.003).The detection rates of the different subgroups were as follows: echogenic kidney33.33%(4/12)> duplex kidney23.81%(5/21)> MCDK 23.53%(8/34)>polycystic kidney disease17.65%(3/17)>hydronephrosis15.80%(9/57)>enlarged and echogenic kidney 9.09%(1/11)>dysplastic kidney7.69%(1/13).6、Among the 31 cases with pathogenic CNVs,there were 10(32.26%,10/31)with numerical abnormalities of chromosomes,11(35.48%,11/31)with microdeletion/microduplication syndromes,11(35.48%,11/31)with nonsyndromic CNVs.What’ more,the mutations of HNF1,AGT and REN were the causes of CAKUT,while NPHP1 and KCNJ1 were considered to be the candidate genes.Conclusion1、We identified 6.37% fetuses with abnormal karyotypes with standard karyotyping and further proved that CAKUT is associated with abnormal karyotypes,especially the cases with other congenital anomalies.2、Karyotyping does not appear to be essential in cases of pyelocaliceal dilatation and unilateral renal agenesis;in cases of isolated MCDK and HDN,however,the indication for karyotyping is debatable.3、Those with unbalanced aberration can also been detected by CMA to make clear origin,size and nature.4、CMA plays an important role in clinical diagnosis of fetuses with CAKUT and normal karyotype with a diagnostic rate of 8.94%.In addition,CMA can identify genes in CNVs: HNF1,AGT,REN,NPHP1 and KCNJ1.5、CMA replaces the need for fetal karyotype in case with one or more major structural abnormalities.However,CMA cannot detect balanced inversions,balanced translocations,or all cases of tissue mosaicism.In addition,not all microarrays can detect triploidy,such as array CGH.6、The risk of chromosomal anomalies will be significantly increased when the fetuses with CAKUT with other congenital anomalies.The risk will be different in different subgroups: echogenic kidney>duplex kidney>MCDK>polycystic kidney disease>hydronephrosis>enlarged and echogenic kidney>dysplastic kidney.7、17q12 microdeletion syndrome,22q11.2 microdeletion syndrome and Williams-Beuren syndrome are the most common syndromes in fetuses with CAKUT.The non-syndromic CNVs : 16q13q24.3 duplication,2q14.3-q24.3duplication,3p26.1microdeletion,4q35.2microdeletion,17p12 microdeletion and 17p13.3 microduplication are the first to be reported in CAKUT patients.8、It is recommended that NGS should be used in the CAKUT cases with VOUS and benign CNVs detected by CMA next.Part II Application of WES investigated fetuses with CAKUT Methods1 、 We recruited 30 fetuses with CAKUT with/without other structural abnormalities detected by ultrasound.Of the fetuses,22 exhibited isolated CAKUT,and 8 were associated with other abnormalities.The parents were seen for prenatal diagnosis at our Prenatal Diagnosis Center between February 2014 and February 2016.For 23 of these families,only the proband(Group 1,77%)underwent WES,and for another 7(Group 2,23%),we performed fetus-mother-father trio WES.2 、 Genomic DNA was extracted from fetuses and their parents using Salting-outing? DNA Blood Mini kits,following the manufacturers’ protocol.3、DNA libraries were prepared using a NEXTflex TM Rapid DNA Sequencing Kit(5144-02)according to the manufacturer’s protocol.The libraries were tested for enrichment by q PCR,and size distribution and concentration were determined using an Agilent Bioanalyzer 2100.4、A Hi Seq2500 sequencer was used for sample sequencing according to the manufacturer’s protocol(version 3,Illumina,Inc.,San Diego,California).Two parallel reactions were performed for each sample.5、The raw image files were processed using bcl2 fastq software(Illumina)for base calling and raw data generation.Low-quality sequencing reads were filtered using a quality score ≥20(Q20).The filtered reads were aligned to the NCBI human reference genome(hg19)using Burrows-Wheeler Aligner.6、BAM files were subjected to SNP analysis,duplication marking,indel realignment,and recalibration using SAMtools and Pindel.All variants of each sample were interpreted by annotation,disease prediction and biological analysis.7、The variants were categorized into 5 types by ACMG: pathogenic,likely pathogenic,significance of uncertain,likely benign and benign.8、Sanger sequencings were performed on fetuses and parents with pathogenic or likely pathogenic varants.Results1、We detected 4 pathogenic variants and 2 incidental variants in 30 CAKUT fetuses.Hence,the true diagnostic rate for WES was 4/30(13.3%).2、the detection rate for isolated CAKUT was only 2/.22(9.1%)and for CAKUT with other abnormalities was 2/8(25%).3、We detected pathogenic variants of four genes,UMOD,NEK8,HNF1 B,and BBS2,and 2 incidental variants,HSPD1 and GRIN2 B.4、The turnaround time for Group 1 was approximately 11-12 weeks.In Group 2(n=7),the turnaround time was approximately 8-9 weeks.Conclusion1、WES is an effective method for the identification of etiology in CAKUT fetuses with an extra detection rate of 13.3%.The findings further confirm that CAKUT is associated with monogenic defects,particularly those with other anomalies and provide the possibility for PGS.2、The findings help guide prenatal care,evaluate the prognosis of fetus and decide the time to terminate the pregnancy.3、The reporting of SFs presents signifcant opportunities to prevent disease,but this process may introduce fiscal and other costs and it will be important for the genomics community to study the impact of SFs on the busy clinician,individual patients and families,and populations at large.4、The numbers in this study were too small to draw useful conclusions regarding the value of testing in fetuses with isolated echogenic kidneys,the utility of WES for echogenic kidneys alone should be questioned.5、Trio WES not only improve the diagnosis rate but also decreased the number of Sanger cosegregation analyses considerably,affected de novo mutation analysis and was able to identify a limited number of variants located in novel candidate disease-causing genes.6、WES was able to expedite interpretation and shorten the turnaround time.Our data lead us to recommend that,ideally,trios should be analyzed simultaneously,although doing so is substantially more expensive than routine clinical WES performed postnatally on a child.