Application of Interphase Fluorescent in Situ Hybridization: a Screening Tool for the Diagnosis of Microdeletion Syndrome.

BACKGROUND: Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy between the results of interphase and metaphase, we compared the quantitative results of FISH for 5 kinds of microdeletion syndrome and gender determination disorders (SDD). METHODS: A total of 282 (135 for DiGeorge syndrome, 20 for Kalmann syndrome, 7 for Miller-Dieker syndrome, 38 for Prader Willi/Angelman syndrome, 62 for Williams syndrome, and 20 for SDD (SRY FISH)) were enrolled. For SRY FISH, we artificially mixed fresh blood of male and female with various ratios and then compared the results of metaphase and interphase SRY FISH. Using a bio-cell chip, we performed interphase FISH in 168 patients with microdeletion syndromes and compared the results with manual interphase. RESULTS: The concordance rate between the results of metaphase and interphase was 100% in microdeletion syndrome. In the disorders of gender development, SRY FISH showed 100% concordance between interphase and metaphase when we counted 50 metaphase cells and 100 interphase cells. Comparison with mixtures of male and female blood at various ratios also showed 100% concordance. The results of bio-cell chip showed 100% concordance between previous interphase FISH results. CONCLUSIONS: Considering the complete concordance between interphase and metaphase in microdeletion syndrome, the application of interphase FISH without performing metaphase FISH can be a screening test for microdeletion syndrome. Confirmation by metaphase FISH can be performed only in cases with abnormal results by interphase FISH.

Molecular diagnosis of hereditary spherocytosis by multi-gene target sequencing in Korea: matching with osmotic fragility test and presence of spherocyte.

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.

Cytogenetic heterogeneity and their serial dynamic changes during acquisition of cytogenetic aberrations in cultured mesenchymal stem cells.

To minimize the risk of tumorigenesis in mesenchymal stem cells (MSCs), G-banding analysis is widely used to detect chromosomal aberrations in MSCs. However, a critical limitation of G-banding is that it only reflects the status of metaphase cells, which can represent as few as 0.01% of tested cells. During routine cytogenetic testing in MSCs, we often detect chromosomal aberrations in minor cell populations. Therefore, we aimed to investigate whether such a minority of cells can expand over time or if they ultimately disappear during MSC passaging. We passaged MSCs serially while monitoring quantitative changes for each aberrant clone among heterogeneous MSCs. To investigate the cytogenetic status of interphase cells, which represent the main population, we also performed interphase FISH analysis, in combination with G-banding and telomere length determination. In human adipose tissue-derived MSCs, 4 types of chromosomal aberrations were found during culturing, and in umbilical cord MSCs, 2 types of chromosomal aberrations were observed. Sequential dynamic changes among heterogeneous aberrant clones during passaging were similar to the dynamic changes observed in cancer stem cells during disease progression. Throughout all passages, the quantitative G-banding results were inconsistent with those of the interphase FISH analysis. Interphase FISH revealed hidden aberrations in stem cell populations with normal karyotypes by G-banding analysis. We found that telomere length gradually decreased during passaging until the point at which cytogenetic aberrations appeared. The present study demonstrates that rare aberrant clones at earlier passages can become predominant clones during later passages. Considering the risk of tumorigenesis due to aberrant MSCs, we believe that our results will help to establish proper safety guidelines for MSC use. In particular, we believe it is critical to test for chromosomal aberrations using both G-banding and FISH to ensure the safety of human stem cells that are manufactured in vitro for clinical applications.