Application Of Chromosome Microarray Analysis (CMA) Investigated33Patients With Congenital Cleft Lip And Palate

Objective1、To explore the value of the application of whole-genome high resolutionchromosome microarray analysis (CMA) investigated33patients with congenitalcleft lip and cleft palate.2、To explore the differences of pathogeneic copy number variants (CNVs) detectionrate among the patients with isolated cleft lip (CL), isolated cleft palate (CP) andcleft lip with cleft palate (CLP).3、Provide a theoretical basis for establishing clinical operation procedures duringthe genetic diagnosis and prenatal diagnosis of congenital cleft lip and cleft palate.4、To explore the strategy to reduce the difficulties of genetic diagnosing andcounseling the variants of unknown significance (VOUS) identified by CMA.Methods1、A total of33patients referred for syndromic and nonsyndromic cleft lip and cleftpalate and their parents were received from the Department of stomatology inGuangzhou Women and Children’s Hospital during August2012through June2013.Twenty-nine of these patients were characterized with nonsyndromic clefts,10ofwhom were CL,11CLP,8CP. Four of them were syndromic clefts.2、Chromosomal analyses were performed on patients’blood using conventionalGTG-banding techniques at the320band level.3、Genomic DNA was extracted from peripheral blood of the patients and theirparents using QIAamp DNA Blood Mini kits (Qiagen, Germany), following themanufacturer’s protocol. The amount of genomic DNA applied to the CytoScanTMArray requires at least250ng. 4、The patients’ DNA samples were run on CytoScanTMHD Array using themanufacturer’s protocol. Array is scanned and generates intensity (CEL) file data.Then the probe level analysis on CEL file data is performed in ChAS software(Affymetrix, California, USA).5、The CNVs detected were further aligned with known CNVs listed in database athome 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 fatherly confirm VOUS werede novo or inherited.7、All pathogenic CNVs identified by CMA were further confirmed by Real-TimeqPCR according to the standard procedures.Results1、A total of33patients were performed on CMA. CNVs identified by CMA in6patients were considered to be pathogenic, the detection rate was18.2%; CNVs in26patients were considered to be benign，the detection rate was78.8%; CNVs in1patient were considered to be VOUS, the detection rate was3.0%.2、CNVs identified by CMA in6patients were considered to be pathogenic. Four ofthese six subjects were with nonsyndromic clefts, including that three were withCPO and the other one with CLP. And the pathogenic CNVs were as followed:10q22.2-q22.3microdeletion (1766kb)，20p12.1microdeletion (184kb)，22q11.21-q11.23microdeletion (3163kb)，8p23.1microduplication(198kb). Two ofthem were with syndromic clefts, and the pathogenic CNVs were18q12.3microduplication（638kb）and6q26microdeletion（389kb）.3、Patient CNVs identified by CMA in26patients were considered to be benign.Nineteen of these26subjects’ CNVs were found in DGV/CHOP Database online,and the most common CNVs found in our study were8p11.2microdeletion/microduplication （11.9%），14q32.33microduplication （18.1%），14q11.2microdeletion（5.8%），22q11.22microduplication（11.9%）. The remaining4patients’ were inherited from one of the normal parents, and the CNVs were9q31.1 microduplication,10p12.33microdeletion,7q31.1microdeletion, Xp22.33microduplication.4、CNVs identified by CMA in1patient were considered to be VOUS, and theCNVs was5q21.1microdeletion（110kb）.Conclusion1、CMA is an effective method for the identification of etiology in cleft patients withan extra detection rate of18.2%and has potential to identify new candidate genesfor congenital cleft lip and palate.2、The detection rate of pathogenic CNVs in sydromic clefts was much higher thanthe detection rate in nonsydromic clefts.3、The detection rates for nonsydromic clefts were isolated CP(37.5%)> isolatedCLP(9.1%)> isolated CL(0). Cases with isolated CL have the most favourableprognosis and do not require conventional karyotyping.4、It is recommended that CMA should be the standard technique to identifychromosomal defects in children with isolated CP and CLP as well as syndromicclefts. This would aid in more optimal genetic diagnosis and prenatal counsellingand timely treatment of children with congenital cleft lip and palate.5、Sufficient communication between technicians and genetic counsellors, referenceto parental testing and comparation with international datas may reduce VOUS.