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Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization.

ABSTRACT: BACKGROUND:Marker chromosomes are small supernumerary chromosomes that cannot be unambiguously identified by chromosome banding techniques alone. However, the precise characterization of marker chromosomes is important for prenatal diagnosis and proper genetic counseling. In this study, we evaluated the chromosomal origin of marker chromosomes using a combination of banding cytogenetics and molecular cytogenetic techniques including diverse fluorescence in situ hybridization (FISH) assays and array comparative genomic hybridization (array CGH). RESULTS:In a series of 2871 patients for whom cytogenetic analysis was requested, 14 cases with small supernumerary marker chromosomes (sSMCs) were identified. Nine sSMCs were mosaic, and five nonmosaic. Of the nine cases with known parental origins, four were identified as de novo, and four and one were maternally and paternally inherited, respectively. Six sSMCs were identified by FISH using centromeric probes; three sSMCs were derived from chromosome 15, including two heterochromatic sSMC(15)s and a large sSMC(15) spanning 15q11.1q13.1, and three sSMCs originated from chromosome 14 or 22. Array CGH revealed two cases with derivatives of chromosome 2 and whole chromosome painting multicolor-FISH (M-FISH) identified three cases with derivatives of chromosome 6, 16, and 19, respectively. One maker chromosome in Turner syndrome was characterized as sSMC(X) by preferential application of a centromeric probe for X-chromosome. In addition, one sSMC composed of genomic materials from chromosomes 12 and 18 was identified in parallel with parental karyotype analysis that revealed the reciprocal balanced translocation. CONCLUSIONS:This report is the largest study on sSMCs in Korea and expands the spectrum of sSMCs that are molecularly characterized.

SUBMITTER: Jang W

PROVIDER: S-EPMC4977864 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization.

Jang Woori W Chae Hyojin H Kim Jiyeon J Son Jung-Ok JO Kim Seok Chan SC Koo Bo Kyung BK Kim Myungshin M Kim Yonggoo Y Park In Yang IY Sung In Kyung IK

Molecular cytogenetics 20160808

<h4>Background</h4>Marker chromosomes are small supernumerary chromosomes that cannot be unambiguously identified by chromosome banding techniques alone. However, the precise characterization of marker chromosomes is important for prenatal diagnosis and proper genetic counseling. In this study, we evaluated the chromosomal origin of marker chromosomes using a combination of banding cytogenetics and molecular cytogenetic techniques including diverse fluorescence in situ hybridization (FISH) assay ...[more]

PMID: 27508004

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Project description:BACKGROUND: In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. Despite the benefits of this technique, visualization of overlapping transcript distributions by differently colored precipitates remains difficult because of masking of lighter signals by darker color precipitates and lack of three-dimensional visualization properties. Fluorescent detection of transcript distributions may be able to solve these issues. However, despite the use of signal amplification systems for increasing sensitivity, fluorescent detection in whole-mounts suffers from rapid quenching of peroxidase (POD) activity compared to alkaline phosphatase chromogenic reactions. Thus, less strongly expressed genes cannot be efficiently detected. RESULTS: We developed an optimized procedure for fluorescent detection of transcript distribution in whole-mount zebrafish embryos using tyramide signal amplification (TSA). Conditions for hybridization and POD-TSA reaction were optimized by the application of the viscosity-increasing polymer dextran sulfate and the use of the substituted phenol compounds 4-iodophenol and vanillin as enhancers of POD activity. In combination with highly effective bench-made tyramide substrates, these improvements resulted in dramatically increased signal-to-noise ratios. The strongly enhanced signal intensities permitted fluorescent visualization of less abundant transcripts of tissue-specific regulatory genes. When performing multicolor fluorescent in situ hybridization (FISH) experiments, the highly sensitive POD reaction conditions required effective POD inactivation after each detection cycle by glycine-hydrochloric acid treatment. This optimized FISH procedure permitted the simultaneous fluorescent visualization of up to three unique transcripts in different colors in whole-mount zebrafish embryos. CONCLUSIONS: Development of a multicolor FISH procedure allowed the comparison of transcript gene expression domains in the embryonic zebrafish brain to a cellular level. Likewise, this method should be applicable for mRNA colocalization studies in any other tissues or organs. The key optimization steps of this method for use in zebrafish can easily be implemented in whole-mount FISH protocols of other organisms. Moreover, our improved reaction conditions may be beneficial in any application that relies on a TSA/POD-mediated detection system, such as immunocytochemical or immunohistochemical methods.

Dataset Information

Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization.

Publications

Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization.

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