The Investigation Of Genetic Etiology Of Unexplained Intellectual Disability/Global Developmental Delay

Background and ObjectiveIntellectual Disability(ID),also known as Developmental Delay(DD),is characterized by obvious limitations of cognitive and social adaptive functions and is diagnosed before 18 years old.DD is used for children younger than 5 years old.The development assessment method is relatively stable and reliable.The diagnostic criteria are children with large sports,fine sports,language,cognition,adaptive behavior,etc Among the developmental areas,there are two or more areas lagging behind,significantly behind children of the same age(less than 2 standard deviations).Severe cases often do not have the ability to take care of themselves and require long-term support from the family and society.The general population incidence of ID/DD is approximately 1%.According to data from multiple medical survey agencies in the United States,ID has the largest economic burden,about $ 1 million per person in a lifetime.People with intellectual disabilities need lifelong rehabilitation support treatment,which lead a heavy psychological and economic burden on the children's family and society.The etiology of ID/DD is still unclear.How to clarify its pathogenesis is also a very urgent research topic.Therefore,the research about ID is a hot topic in the fields of neuroscience and genetics.The etiology of ID is complicated,both genetic and environmental factors,non-genetic factors account for about 10%.With the improvement of living standards and the improvement of medical and health care measures,intellectual disability caused by factors such as exogenous infections,poisoning,trauma,hypoxia,malnutrition,cultural backwardness,and psychological damage has been greatly controlled,and genetic factors have contributed to prominent.Genetic factors include chromosomal abnormalities,copy number variants,single gene abnormalities,and epigenetic abnormalities.At present,the genetic methods for diagnosing ID mainly include chromosome karyotype analysis and fluorescence in situ hybridization(FISH).In recent years,it has been found that copy number variation is closely related to ID It is possible to analyze CNVs across the genome.Chromosomal Microarray Analysis(CMA).These include Array Comparative Genomic Hybridization(array-CGH)and Single Nucleotide Polymorphisms Microarrays(SNP-array).However,none can detect changes in chromosomal balance,such as point mutations.With the rapid,rapid,and sensitive development of the next-generation technology,the process of diagnosing unexplained intellectual disability has transitioned to WES(whole exome sequencing,WES)through CNV analysis.Human exons account for 1-2%of the entire genome-encoded protein sequence,but cover 85-90%of the encoded genes.Whole exome sequencing can be used to detect disease-causing genes related to known diseases and to discover new disease-related genes.At present,there are not many reports of ID gene mutations in our country.Our research uses WES technology to sequence and analyze ID patients to detect CNVs related to ID.In this study,we used CMA and WES technology to detect children with unexplained ID/DD or other abnormalities,including 569 cases of CMA received first-line testing and 146 cases was further tested by WES because of the normal results of karyotype and CMA analysis.aiming at:1)To explore the genetic causes of children with unexplained ID/DD from the genomic and single gene levels.2)Evaluate the recurrence risk of ID/DD families to provide a basis for whether prenatal diagnosis or pre-implantation diagnosis is required for subsequent pregnancy.3)Identify potential copy number variants and candidate disease-causing genes of ID/DD through CMA and WES technology and improve the understanding of ID/DD etiologyPart I Application of CMA investigated children with unexplained ID/DDMethods1.A total of 569 cases of ID/DD children and their parents were diagnosed in the Neurorehabilitation Department of Guangzhou Women and Children's Medical Center during May 2012 to December 2017.ID/DD divided into three groups according to whether other abnormalities were combined.There were 419 cases in the simple ID/DD group,53 cases in the ID/DD combined epilepsy group,and 97 cases in the ID/DD combined with other structural deformities group.All families received genetic counseling and signed informed consent before conducting genetic testing.This study also received approval from the hospital ethics committee.2.The fetal and parent samples are processed according to the standard experimental operating procedure of the American Affymetrix company's CytoScan HD chip platform(1.95 million copy number probes and 750,000 SNP probes).3.Use the matching CHAS software to analyze the data of the.CEL file generated by the scanning chip.4.According to DGV(including CNVs of normal people),DECIPHER(including phenotype and pathogenic fragments of patients),OMIM(including known pathogenic genes),CAGdb,ISC A(including benign and pathogenic CNVs),UCSC Genome Browser(displaying the content and function of the genes in the fragments),PUBMED,etc.and the internal database of the laboratory to compare the analysis results online to judge the nature of CNVs.5.Both of the parental DNA was detected by CMA to further confirm pathogenic CNVs and VOUS were de novo or inherited.6.Chi-square test for statistical analysis(SPSS 22.0)Results1.CM A was used as a first-line detection technique to detect 569 children with unexplained ID/DD with or without other abnormalities.Pathogenic CNVs were detected in 162 children,and the detection rate of pathogenic CNVs was 28.5%(162/569).The detection rate of VOUS was 2.6%(15/569),while the detection rate of benign CNVs in children with DD/ID was 68.9%(392/569).2.The detection rate of pathogenic CNVs in the simple ID/DD group was 26.5%;the ID/DD combined with epilepsy group was 26.4%;the ID/DD combined with other structural malformations group was 38.1%.Chi-square test was used for pairwise comparison.?=0.05 was the test level.The results showed that there was a statistically significant difference in the detection rate of pathogenic CNVs between the DD/ID group and the DD/ID group combined with other structural deformities(P=0.02,26.5%vs 38.1%,x2 test).There was no statistically significant difference between the other groups.3.Of the 162 cases of fetuses with pathogenic CNVs,6 had abnormal chromosome numbers,accounting for 3.7%(6/162).85 known cases of microdeletion/microduplication syndrome,accounting for 52.5%(85/162);82 of them were common microdeletion/microduplication syndrome,including Angelman/Prader-Willi syndrome,Williams-Beuren syndrome,22q11.2 microdeletion/microduplication syndrome,Wolf-Hirschhom syndrome,16p 11.2 microdeletion/microduplication syndrome,1p36 microdeletion syndrome,17p11.2 microdeletion/microduplication syndrome,5P deletion syndrome,2q33.1 deletion syndrome,2q37 monomer syndrome,Rubinstein-Taybi syndrome,Silver-Russell syndrome;there are 3 other rare microdeletion/microduplication syndromes,including 15q24 microdeletion syndrome,Xq28 microduplication syndrome As well as ophthalmic,cerebral and renal syndromes.4.The remaining 71 cases(43.8%,71/162)are newly-developed non-syndromic pathogenic fragments.Among them,ABAT and PMM2 genes in 16p13.2 region,FTSJ1 gene in Xp11.23p11.22 region,DYNC1H1 gene in 14q32.31q32.33 region and SETBP1 gene in 18q12.3q21.1 region are new pathogenic factors for ID/DD Candidate genes.Conclusion1.In this study,whole-genome high-resolution CMA technology was used to detect children with unexplained ID/DD,and the total pathogenicity detection rate was as high as 25.8%.It proves the important value of CMA in clinical application.2.52.5%of the clinically significant CNVs detected in this study are known microdeletions/microduplication syndromes.Angelman/Prader-Willi syndrome was the most common.Another 43.8%were de novo non-syndromic pathogenic fragments,enriching the microdeletion/microduplication disease spectrum of ID/DD.3.CMA has the ability to identify new ID/DD pathogenic candidate genes:involving ABAT,PMM2,FTSJ1,DYNC1H1 and SETBP1 genes.Part ? Application of WES investigated children with unexplained ID/DDMethods1.Select clinical data of ID/DD children and their parents were diagnosed in the Neurorehabilitation Department of Guangzhou Women and Children's Medical Center during May 2018 to October 2019.146 cases with no abnormalities in karyotype and CMA results of the core pedigree of children with ID/DD.Children with ID/DD were divided into three groups according to whether they were combined with other abnormalities,including 77 cases in the simple ID/DD group,29 cases in the ID/DD combined epilepsy group,and 40 cases in the ID/DD combined with other system abnormalities group.All families received genetic counseling and signed informed consent before conducting genetic testing.This study also received approval from the hospital ethics committee.2.A HiSeq2500 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.3.The raw image files were processed using bcl2fastq software(Illumina)for base calling and raw data generation.The filtered reads were aligned to the NCBI human reference genome(hg19)using Burrows-Wheeler Aligner.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.4.The variants were categorized into 5 types by ACMG:pathogenic,likely pathogenic,significance of uncertain,likely benign and benign.5.Sanger sequencings were performed on fetuses and parents with pathogenic or likely pathogenic varants.Results1.Trio-WES was tested the core family samples of 146 children with unexplained ID/DD negative in karyotype and CMA results.Pathogenic/likely pathogenic mutations were found in 61 children,including 51 known pathogenic genes and 64 mutation sites related to the phenotype of children.Therefore,The overall molecular diagnosis rate for WES in ID/DD children with Mendelian single gene disease was 41.78%(61/146).2.Among the 146 ID/DD children who received WES testing,the positive mutation detection rate in the simple ID/DD group was 36.36%(28/77);the detection rate in the ID/DD combined epilepsy group was 41.38%(12/29);the detection rate of ID/DD combined with other system abnormal groups was 52.5%(21/40).Chi-square test was used for multiple comparison,the test level was ?=0.05,and there was no statistically significant difference in the detection rate of positive mutation among groups.3.We detected pathogenic/likely pathogenic mutations in 61 children with ID/DD,including 51 known pathogenic genes.Eight of the disease-causing genes occur more frequently in children(Table 4),including the genes MECP2,ATRX,ADNP,SOX11,AHDC1,ARID1B,SLC9A6,and BRAF.The two most frequent are the MECP2 gene(n=4)and the ATRX gene(n=3),which lead to Rett syndrome and X-linked mental retardation-hypertensive syndrome type 1 respectively.4.WES found 64 positive mutation sites in 61 children with ID/DD.According to Mendelian inheritance,it included 40 cases of children with autosomal dominant inheritance(AD)sites(n=40),and 6 cases(including 2 cases of UPD origin)with autosomal recessive inheritance(AR)sites(n=9),8 cases contained X chromosome dominant inheritance(XD)sites(n=8)and 7 cases contained X chromosome recessive inheritance(XR)sites(n=7).According to the source of the mutation site,there were 47 children with new mutations(n=47)and 14 children with hereditary mutations(n=17).Conclusion1.In this study,We conducted WES testing for children with ID/DD and the total positive detection rate was 41.78%,which further confirmed the application value of WES technology in children with ID/DD.The findings provide genetic basis for the early diagnosis of diseases,precise treatment and prognosis evaluation.2.Among the ID/DD-positive cases in this study,the most relevant genes are MECP2?ATRX?ADNP?SOX11?AHDC1?ARID1B?SLC9A6 and BRAF?3.Among the positive cases in this study,most of cases were de novo and a small proportion inherited from parents;Most of children had a lower risk of recurrence,and had a higher risk of recurrence;It provide a scientific basis for genetic counseling and fertility guidance for these families.4.For UPD in non-imprinted chromosomes,it is necessary to consider the risk of recessive inherited diseases.