Clinical Cytogenetic Diagnosis Research Articles

Analysis of Xp22.31q27.1 Region Variation Detection based on SNP Array Technology.

The aim of the study was to investigate the value of single nucleotide polymorphism array (SNP array) technology in the prenatal diagnosis. The variants in Xp22.31q27.1 region of 13 fetuses were analyzed by SNP array technology. Chromosome karyotype analysis was also performed for these fetuses and their parents. Chromosome karyotype analysis found no obvious chromosomal abnormality at 400 bands resolution. Using SNP array technology, we found that all fetuses had mutations in Xp22.31q27.1 region, which was mainly Xp22.31 lesion (61.5%, 8/13) that contained 2 to 5 OMIM genes. Moreover, these mutations consisted of 7 cases of repetition and 6 cases of deletion. In addition, 9 variants were inherited from their mothers, 3 mutations were inherited from the father, and 1 variant was de novo. Compared to traditional analysis of chromosome karyotype, SNP-array technology can detect more chromosomal microdeletions and microduplications. SNP-array technology can act as a supplementary diagnostic method in clinical cytogenetic diagnosis.

Targeted Microarray Analyses Augment the Clinical Cytogenetic Diagnosis of Acute Lymphoblastic Leukemia (ALL): Submicroscopic Genetic Events Improve Diagnosis, Contribute to Risk Stratification, and Provide Genetic Markers for Minimal Residual Disease (MRD) Testing

AbstractAbstract 2690Cytogenetic aberrations are key diagnostic and prognostic markers in ALL; however, suboptimal chromosome morphology, low lymphoblast mitotic activity in cell culture and the inability to detect subtle (≤ 5∼10 Mb) abnormalities often preclude a rapid and accurate ALL clinical cytogenetic workup. Recent genome-wide microarray studies have reported submicroscopic DNA copy number alterations (CNAs) in ALL that target key genes involved in B-cell and T-cell cellular processes (cell cycle regulation, differentiation, proliferation and survival) and alterations of these genes are rapidly being associated with clinical treatment and outcome (e.g., IKZF1, NUP214/ABL1, NUP214/SET, PTEN, PTPN2). To improve the cytogenetic diagnosis of ALL, we analyzed DNA samples from 22 newly-diagnosed, pediatric T-cell and 22 B-cell (16 newly diagnosed and 6 relapse) ALL patients (pts) using a 133K targeted oligo-based microarray. The aCGH results were compared to their cytogenetic, FISH and clinic-pathological findings. When sufficient material was available, locus-specific FISH studies were performed to confirm the submicroscopic CNAs. The 22 pediatric T-cell samples, obtained from the COG Cell Bank, showed the following karyotypes: del(6q) alone (n=10), del(6q) with one or two additional abnormalities (n=8), or normal karyotypes (n=4). The B-cell ALL karyotypes showed one to three abnormalities (n=10), ≥5 abnormalities (n=10), including hypodiploidy/hyperdiploidy/near-triploid/near tetraploidy, or normal karyotypes (n=2). Twelve B-cell ALL pts showed prognostically-significant translocations. By aCGH, CNAs were observed in all 44 cases, ranging from five to14 (median, 8) CNAs in T-cell and three to 35 (median, 10) CNAs in B-cell ALL. aCGH detected del(6q) in all 18 known T-cell pts (size range, 16.8 kb to 106 Mb) and in six B-cell pts. Submicroscopic aberrations detected in T-cell ALL included: CDKN2A (mono or bi-allelic) deletions (n=19), ranging in size from 24 kb to 6.76 Mb, including 6 focal deletions under 200 kb, a ∼77 Kb deletion in 1p33, resulting in a STIL/TAL1 fusion (n=8), other TAL1 or STIL deletions (n=3), PTEN deletions (n=6) ranging from 15 kb to 1 Mb (n=6), 49 kb biallelic GSTT1 deletions (n=4), and TLX3 rearrangements (n=2) including a case with a ABL1/NUP214 fusion and a focal biallelic PTPN2 deletion. Cryptic deletions in 4q31.3/FBXW7 and in 9q34 resulting in a SET/NUP214 fusion and duplication of MYB were observed in a single case. In B-cell ALL, recurring “cryptic” deletions were of IKZF1 (n=6), TOX (n=3), PAX5 (n= 5) CDKN2A (mono- or bi-allelic) (n=10), ETV6 (n=6), BTG1 (n=4), C20orf94 (n=3), EBF1 (n=2), TP53 (n=2), and miR650 (n=2). Single CNAs observed in B-cell ALL included: CDKN2C, LCK, BTLA, MECOM, TBL1XR1, AFF1, LEF1, HEF, RAG1, ATF7IP, JAK2, PTEN, ID4, CASC3, COMMD1, and the drug receptor gene NRCC1. Gains observed in residual or relapsed B-cell ALL were MYC, MLL, miR657, and at 13q31.3, which includes GPC5 and multiple miRNAs (latter also seen in three cases of T-cell), and PPP2R5A in a t(4;11) pt. Imbalances in TRG (7p14), TRB (7q34), and TRA/TRD (14q11.2) were noted in T-cell pts whereas IGH, IGK and/or IGL were clearly seen in B-cell pts. Three translocations, t(9;22), t(4;11) and t(12;21), were also detected by microarray using linear amplification followed by aCGH. These findings demonstrated that aCGH can improve the cytogenetic diagnosis of ALL, contribute to risk stratification, and provide genetic markers for MRD testing. Moreover, these results provide a rationale for the integration of targeted microarray technology in the clinical evaluation of ALL. Disclosures:Slovak:PerkinElmer: Employment. Hunger:bristol myers squibb: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees. Shaffer:PerkinElmer: Employment; American College of Medical Genetics Foundation: Membership on an entity's Board of Directors or advisory committees. Ballif:PerkinElmer: Employment. Schultz:PerkinElmer: Employment.

Cytogenetic and molecular characterization of partial trisomy 4q and partial monosomy 10q in a patient

To ascertain the karyotype of a girl with moderate mental retardation and growth retardation, perform correlation analysis between chromosomal variation and phenotype, and investigate the application and superiority of array-based comparative genomic hybridization (array-CGH) in clinical cytogenetic diagnosis. G-banded chromosome analysis, array-CGH, fluorescence in situ hybridization (FISH) and real-time quantitative PCR (RQ-PCR) were used to ascertain the karyotype of the patient and her relatives. G-banding analysis of the patient showed a derivative chromosome 10 with an extra fragment on its long arm terminal, both her father and grandmother had an apparently balanced translocation t(4;10)(q25;q26). Array-CGH revealed that the breakpoint on chromosome 4 was located at 4q26. In addition, a microdeletion of about 0.54 Mb del(10)(q26.3) was identified from the patient. FISH and RQ-PCR confirmed that the del(10)(q26.3) was also present in both her father and grandmother. No recognizable phenotype was associated with del(10)(q26.3). The abnormal phenotypes presented in the patient may be ascribed to the 4q26-q35.2 triplication. Further more, compared with conventional cytogenetic analysis, array-CGH is of high resolution and high accuracy.

Validation of the Agilent 244K Oligonucleotide Array–Based Comparative Genomic Hybridization Platform for Clinical Cytogenetic Diagnosis

High-resolution microarray comparative genomic hybridization (aCGH) is being adopted for diagnostic evaluation of genomic disorders, but validation for clinical diagnosis has not yet been reported. We present validation data for the Agilent Human Genome Microarray Kit 244K for clinical application. The platform contains approximately 240,000 distinct 60-mer oligonucleotide probes spanning the entire human genome. We studied 45 previously characterized samples (43 abnormal, 2 normal), 32 with knowledge of prior results and 13 in a blinded manner with 11 performed in a reference laboratory providing microarray testing. Array analysis confirmed known aberrations in 43 samples and a normal result in 2. The array analysis corrected 1 karyotype and clarified 2 additional cases. Array data from 6 patients with 22q11.2 deletion found an average of 2.56 megabases (Mb; range, 2.49-2.62 Mb) with a common 2.43-Mb deleted region. Approximately 7 copy number variants from 400 base pairs to 1.6 Mb were identified per sample. Results demonstrate the usefulness of the aCGH-244K platform as a powerful diagnostic tool.

Copy number variations and clinical cytogenetic diagnosis of constitutional disorders

The recent appreciation of widespread copy number variation in the genomes of healthy human beings has presented a significant challenge to clinical cytogeneticists who wish to use genome-wide array comparative genomic hybridization (CGH) assays for clinical diagnostic purposes. Clinical cytogeneticists need to differentiate between copy number variants (CNVs) that are likely to be pathogenic and CNVs that are less likely to contribute to an affected individual's clinical presentation. Unfortunately, our knowledge of the phenotypic effects of most CNVs is minimal, leading to the classification of many CNVs as genomic imbalances of unknown clinical significance. This has caused many laboratories to resist the use of higher-resolution genome-wide array CGH assays for clinical purposes. Ironically, the accumulation and annotation of such array CGH data can lead to the rapid identification of pathogenic CNVs and the definition of new genomic syndromes that, in turn, are useful for accurate clinical genetic diagnoses.

Development and validation of a CGH microarray for clinical cytogenetic diagnosis

PurposeWe developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). MethodsThe array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference. ResultsThe CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%. Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory. ConclusionThe array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype. Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.