The Application Of Array Based Comparative Genomic Hybridization In Diagnosis And Prenatal Diagnosis The Imbalanced Chromosomal Aberration

Partial deletion and duplication are common unbalanced chromosomal aberrations and often associated with multiple congenital anomalies, developmental delay and mental retardation. Although a conventional cytogenetic analysis by G-banding can detect numerical aberrations and some apparent structural aberrations, it cannot detect deletions or duplications smaller than 5 Mb. It is possible to confirm or detect the suspect micro-deletions or duplications by fluorescence in situ hybridization (FISH). However, without any of the indicative chromosomal aberration information, FISH is not feasible. Even though the 24 fluoresencetly labeled chromosomal painting probes utilized by spectral karyotypin (SKY) can detect the origin of the apparent translocations and marker chromosomes, SKY is impossible to detect the aberrations within a chromosome. Array-based comparative genomic hybridization (array-CGH or aCGH) is a recently developed molecular karyotyping technology permiting the simultaneous rapid high resolution genome-wide copy number variants (CNVs) analysis and mapping of DNA sequences by a hybridization experiment. Since developed, it is admitted to be the most effective tool for detecting chromosome microdeletions and microduplications. Array-CGH technology can improve the diagnostic detection rate of these small chromosomal abnormalities with novel high-resolution, whole-genome screening efficiency and assist clinical referring doctors to precise evaluate patients' condition and their prognosis base on the genotype-phenotype relation analysis. Our current study is the first investigation of application aCGH technology in diagnosis and prenatal diagnosis of unbalanced chromosome aberration in mainland China. Objection6. Establishing the technical system of array-based comparative genomic hybridization (array-CGH), and to study the application of array-CGH in the unbalanced chromosomal aberration.7. To study the sensibility and acccuracy of array-CGH to diagnosis the unbalanced chromosomal aberration.8. To investigate how to combination the technical of array-CGH, G-banding, FISH, C-banding and silver-staining in the diagnosis of unbalanced chromosomal aberration, and to build up an exercisable standard clinical procedure.Subjects and Methods1. SubjectsDuring Jan. 2008 to Dec. 2009, it selected 21 cases of chromosome analysis that came from the genetic clinic of Guangzhou women and children medical centre or other hospitals, including 19 cases of peripheral blood sample, 1 amnionic fluid and 1 fetal cord blood sample.Among 21 cases, there were 4 cases of normal controlled genome, the remaining cases were suspected of unbalanced chromosome abnormalities, but the conventional G-banding could not confirm the diagnosis.2. Method(1) All of 21 DNA samples were performed according to the standard operation procedure of SNP6.0 chip (Affimetrix) , and were analyzed by the matching scanner and software of computer. (2) Using PCR or FISH to confirm the array-CGH results to assess the accuracy and sensibility of array-CGH in diagnosis the unbalanced chromosome aberration.(3) Using C-banding and silver staining technology to confirm the duplication occurred in heterochromatin and satellite region to evaluate the prognosis.(4) To study the effect in all kinds of banding techniques and analysis techniques of chromosomes, and to build up an standard clinical procedure.3. follow-up We made routine postnatal follow-up for the prenatal cases.Results1. All of 21 samples were successful detected by array-CGH technology, including which 17 patient cases that can not diagnosed by the conventional G banding were all precise identified at molecular level.2. Case 1: There was some abnormal at the long arm of chromosome 13 [the karyotype of G-banding is 46,XY, 13q-? ]. The result of array-CGH is 46,XY, del(13)(q33.2â†'q34). The size of the deletion is 8.5Mb.3. Case 2, 4 and 6 : They were all found to have some part of extra material on chromosome 18 and chromosome 9, respectively, which the source were not determined by routine G-banding. Case 6 is the father of case 4. The array-CGH result of case 2 is 46,XY,dup(18)(q12.3â†'q22.3), the size of duplication is 34 Mb. There was a inherited duplication on the long arm of chromosome 9 at case 4 transmitted from her father. The array-CGH result is 46,XX,dup(9)(9p13â†'q13),pat. The size of the duplication is 30 Mb.4. Case 3 and case 5 : There are the cases of complex genomic duplications and deletions. Using array-CGH technique identified the karyotype of 46,XY,dup(8) (p21.3-p11.3),de1(8) (p23.3â†'p23.1) in case 3. The size of duplication and deletion are 15.8 Mb and 9.1 Mb, respectively. The array- CGH result of case 5 is 46,X,dup(X)(q21.31â†'q28),del(X) (p22.33â†'p11.22) which including 63.45 Mb duplication on the long arm and 52.7 Mb deletion on the short arm of chromosome X.5. Case 7—10 are female patients with normal karyotype by the conventional G-banding analysis. Array-CGH identify that they all have a micro-deletion on the long arm of chromosome 17 with the size of 380 kb [46 , XX ,del(17)(q21.31-q31.32) ].6. The situation of case 11-15 is similar to case 7—10, however, the submicroscopic deletion occur on their terminal short arm of chromosome X with the size of 112 kb[46,XX,del(X)(p22.33)].7. Case 16 and Case 17 are the cases of prenatal diagnosis. There is no visible abnormality by routine G-band analysis in case 16, but a de novo microdeletion with the size of 500 kb was found on the chromosme 22[46,XN,del(22)(q11)]. By taking account of the malformation in the fetal cardiac ultrasonography, we consider it is DiGeorge syndrome. The pregnancy is terminal because DiGeorge syndrome will lead to mental retardation. In case 17, the balanced translocation is genetic from pregnancy women whose phenotype is normal. Array-CGH doesn't identify any large-scale pathogenic DNA copy number variation. It can predict that fetal's phenotype will be the same with the mother and suggest that pregnancy can continue. The baby was born in term, and she is normal.8. All of the results above are confirmed by PCR or FISH technology, and the confirmed result are agree with that of array-CGH.Conclusion1. 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. array-CGH directly hybridize the sample DNA with human genome nucleotide probes on the micro-array and then screening the whole genome and mapping of DNA sequences more accurate and more rapid.3. Array-CGH can be used in many clinical questional chromosome disease. It can assist clinical doctors to precise evaluate patients' condition and their prognosis and guide the pregnancy outcome base on the genotype-phenotype relation analysis.4. Although array-CGH has powerful efficiency in identifying copy number variations (CNV), it has some limitations too: Firstly, it can not direct diagnosis the aberration without CNV, such as balanced inversion. Secondly, genetics workers need to be careful to determine whether the result of array-CGH is pathogenic or polymorphic variation.5. To diagnosis the unbalanced chromosomal aberration, we recommend the process of karyotype analysis: using G-banding to screening first; and array-CGH was selected to diagnosis; FISH or SKY to final confirm the balanced translocation. C-banding and silver-staining technique can help to verify the CNV occurred at heteochromatin and satellite region, and profit to assess the prognostic.