The Application Of Array-based Comparative Genomic Hybridization In Birth Defects

The First Part:Microdeletion and Microduplication Analysis of Chinese Conotruncal Defects Patients with Targeted Array Comparative Genomic HybridizationSection 1:Use cases with known etiology to validate targeted array-CGHObjective:The aim of the study was to validate the performance of Agilent targeted array-CGH by comparing the results of 12 known cases which have diagnosed chromosomal defects.Method:We selected serum of 12 patients, which 10 cases of VCFS have been initially characterized by MLPA, and 2 cases of cri du chat syndrome have been initially characterized by Cytogenetic Whole-Genome 2.7M array, they are all detected again by Agilent targeted array-CGH. Agilent Genomic Workbench Lite Edition 6.5 software is used to analysis the results.Result:In 12 cases, targeted array-CGH detected concordant results with the MLPA and the whole genome chip, among them,8 of VCFS patients have 22q11.2 microdeletion,2 of VCFS patients have 22q11.2 microduplication, while two of Cri du chat syndrome patients have partial deletion of 5q.Conclusion:The 8x15K targeted array-CGH can rapidly and accurately detect the 22q11.2 microdeletions or 22q11.2 microduplication syndrome, Cri du chat syndrome and other eleven kinds of syndromes, thus providing a reliable genetic information to relevant person.Section 2:22q11 CNVs analysis with targeted array-CGH in 27 patients withCTDsObjective:The purpose of this experiment is to analyze the genetic etiology of 27 patients with CTDs, especially pathogenic CNVs in 22q11.2 region and its probability of occurrence, this provide a theoretical basis for the next genetic counseling, risk assessment, follow-up treatment.Method:Choose 27 patients with simple CTDs, using gDNA extraction kit (QIAGEN, Germany) to extract gDNA, using Agilent 8×15K targeted array-CGH method to detecte CNVs of chromosome 22q11.2 region, employing Real-time PCR with ViiA 7 Real-time PCR instrument(Applied Biosystems, German) to validate the results of the targeted array-CGH.Result:In 27 patients with CTDs, targeted array-CGH detected one case of 22q11 microdeletion, three cases of 22q11 microduplication. Real-time PCR results are consistent with targeted array-CGH.Conclusion:Test results of 8×15K targeted array-CGH in patients with CTDs,14.8% (4/27) of patients get a clear etiology, of which 3.7%(1/27) is microdeletions,11.1% (3/27) is microduplication. This targeted array-CGH as a fast and accurate new means to study chromosomal disease, there is great significance in the diagnosis of the etiology of congenital malformations.CTDsObjective:The purpose of this experiment is to analyze the genetic etiology of 27 patients with CTDs, especially pathogenic CNVs in 22q11.2 region and its probability of occurrence, this provide a theoretical basis for the next genetic counseling, risk assessment, follow-up treatment.Method:Choose 27 patients with simple CTDs, using gDNA extraction kit (QIAGEN, Germany) to extract gDNA, using Agilent 8×15K targeted array-CGH method to detecte CNVs of chromosome 22q11.2 region, employing Real-time PCR with ViiA 7 Real-time PCR instrument(Applied Biosystems, German) to validate the results of the targeted array-CGH.Result:In 27 patients with CTDs, targeted array-CGH detected one case of 22q11 microdeletion, three cases of 22q11 microduplication. Real-time PCR results are consistent with targeted array-CGH.Conclusion:Test results of 8×15K targeted array-CGH in patients with CTDs,14.8% (4/27) of patients get a clear etiology, of which 3.7%(1/27) is microdeletions,11.1% (3/27) is microduplication. This targeted array-CGH as a fast and accurate new means to study chromosomal disease, there is great significance in the diagnosis of the etiology of congenital malformations.The Second Part:Unbalanced Chromosome Aberration Analysis of Abdominal Wall Defect with Array-based Comparative Genomic HybridizationBackground:Chromosome deletion or duplication is one of the common unbalanced chromosome aberrations, it related to birth defect, pediatric developmental delay, mental retardation and other diseases. Traditional methods have many shortcomings, such as low chromosome resolution, low throughput, can’t detect unknown chromosomal abnormalities. Array-based comparative genomic hybridization (array-CGH) is a highly efficient technique of molecular karyotyping, it can be used to detect genome-wide DNA copy number variants (CNVs) highly-throughput and highly-resolution. And array-CGH can accurately locate a particular DNA sequence variation on chromosome; therefore it is best suited to diagnose chromosome deletions or duplications. At present, many countries have been able to use array-CGH technology to diagnosis and prenatal diagnosis of unbalanced chromosome aberration which caused birth defects. This experiment is about the use of array-CGH technology to detect genomic copy number variants of abortion fetal with abdominal wall defects.Objective:Gastroschisis and omphalocele are two common but different pathogenesis of abdominal wall defects deformity. This study was designed to explore the possible new or same genomic copy number variants in these two types of diseases with array-CGH, and explore the application value of the array-CGH technology in the diagnosis and prenatal diagnosis.Method:Choose 5 cases of gastroschisis and 5 cases of omphalocele abortion fetal tissue samples, using gDNA extraction kit (QIAGEN, Germany) to extract gDNA, using Agilent 244K array-CGH method to detecte CNVs, employing Real-time PCR with ViiA 7 Real-time PCR instrument(Applied Biosystems, German) to validate the results of array-CGH.Result:In 5 cases of gastroschisis, array-CGH found no CNVs. In 5 cases of omphalocele, one case has no CNVs; one case was trisomy 18; No.562 fetal existed 4 chromosomal deletions, was del(5) (q13.1-13.2), del(10)(q21.3-22.1) del(14)(q13.1-13.2) respectively, del(14)(q21.3-22.1); No.569 fetal existed 4 chromosomal deletions, was dup(4)(pl6.3-16.1), dup(7)(p22.1), dup(8)(q24.3), dup(9)(q34.3) respectively; No.598 fetal existed 6 chromosomal deletions, was dup(2)(p22.1), dup(2)(q31.2), dup(2)(q34), dup(3)(q28-29), dup(10)(q21.3), dup(22)(ql2.3) respectively. Two fetal existed same CNVs, that was 10q21.3. Real-time PCR results are consistent with array-CGH.Conclusion:1. Microarray-based comparative genomic hybridization (array-CGH) is a recently developed technology with novel high resolution, high accuracy and more rapid features. It is a major breakthrough to identify the genomic aberration at molecular level. Array-CGH can make up the defect of other technology.2. The pathogenesis of gastroschisis has small relationship with abnormal chromosomal aberrations.3. The pathogenesis of omphalocele has big relationship with abnormal chromosomal aberrations, but didn’t find same pathogenic CNVs, prenatal identification of omphalocele should alert chromosomal abnormalities, and prompt thorough cytogenetic investigations and genetic counseling.