title: A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease.
abstract: BACKGROUND
  Copy number variations (CNVs) of chromosomal region 22q11.2 are associated with a subset of patients with congenital heart disease (CHD). Accurate and efficient detection of CNV is important for genetic analysis of CHD. The aim of the study was to introduce a novel approach named CNVplex®, a high-throughput analysis technique designed for efficient detection of chromosomal CNVs, and to explore the prevalence of sub-chromosomal imbalances in 22q11.2 loci in patients with CHD from a single institute.
  
  
  RESULTS
  We developed a novel technique, CNVplex®, for high-throughput detection of sub-chromosomal copy number aberrations. Modified from the multiplex ligation-dependent probe amplification (MLPA) method, it introduced a lengthening ligation system and four universal primer sets, which simplified the synthesis of probes and significantly improved the flexibility of the experiment. We used 110 samples, which were extensively characterized with chromosomal microarray analysis and MLPA, to validate the performance of the newly developed method. Furthermore, CNVplex® was used to screen for sub-chromosomal imbalances in 22q11.2 loci in 818 CHD patients consecutively enrolled from Shanghai Children's Medical Center. In the methodology development phase, CNVplex® detected all copy number aberrations that were previously identified with both chromosomal microarray analysis and MLPA, demonstrating 100% sensitivity and specificity. In the validation phase, 22q11.2 deletion and 22q11.2 duplication were detected in 39 and 1 of 818 patients with CHD by CNVplex®, respectively. Our data demonstrated that the frequency of 22q11.2 deletion varied among sub-groups of CHD patients. Notably, 22q11.2 deletion was more commonly observed in cases with conotruncal defect (CTD) than in cases with non-CTD (P<0.001). With higher resolution and more probes against selected chromosomal loci, CNVplex® also identified several individuals with small CNVs and alterations in other chromosomes.
  
  
  CONCLUSIONS
  CNVplex® is sensitive and specific in its detection of CNVs, and it is an alternative to MLPA for batch screening of pathogenetic CNVs in known genomic loci.
authors.affiliation: Department of Laboratory Medicine, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, Peoples Republic of China. qingxiao18@163.com.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Pediatric Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, Peoples Republic of China. xuyj1006@126.com.
authors.forename: Yuejuan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Genesky Diagnostics (Suzhou) Inc, Suzhou, Peoples Republic of China. liudeyuan@geneskies.com.
authors.forename: Deyuan
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of Laboratory Medicine, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, Peoples Republic of China. giliangj@163.com.
authors.forename: Juan
authors.initials: J
authors.lastname: Geng
authors.affiliation: Department of Pediatric Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, Peoples Republic of China. chengsun@hotmail.com.
authors.forename: Sun
authors.initials: S
authors.lastname: Chen
authors.affiliation: Genesky Diagnostics (Suzhou) Inc, Suzhou, Peoples Republic of China. zhengwenj@geneskies.com.
authors.forename: Zhengwen
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: Department of Laboratory Medicine, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, Peoples Republic of China. qfu@shsmu.edu.cn.
authors.forename: Qihua
authors.initials: Q
authors.lastname: Fu
authors.affiliation: Department of Pediatric Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, Peoples Republic of China. drsunkun@xinhuamed.com.cn.
authors.forename: Kun
authors.initials: K
authors.lastname: Sun
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D002872:Chromosome Deletion; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics*; D015331:Cohort Studies; D056915:DNA Copy Number Variations / Q000235:genetics*; D056426:Genetic Loci / Q000235:genetics; D006330:Heart Defects, Congenital / Q000235:genetics*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis