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Molecular Diagnosis of Cutaneous Diseases

Karan K. Sra, MD; Michelle Babb-Tarbox, BS; Sina Aboutalebi, BS; Peter Rady, MD, PhD; Gregory L. Shipley, PhD; Dat D. Dao, PhD; Stephen K. Tyring, MD, PhD, MBA
Arch Dermatol. 2005;141(2):225-241. doi:10.1001/archderm.141.2.225.
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Objectives  To provide an update on the molecular procedures used increasingly in the study and diagnosis of a variety of dermatologic malignancies and inflammatory disorders and to explore the potential use of these techniques in clinical dermatology. Herein, we review assays such as G-banding, fluorescence in situ hybridization, comparative genomic hybridization, and spectral karyotyping in conjunction with the polymerase chain reaction and DNA microarrays.

Data Sources  PubMed was searched for published articles on molecular diagnosis and dermatologic diseases.

Study Selection  All English-language studies were selected if they provided useful methodologic information or highlighted the usefulness of molecular techniques.

Data Extraction  Only methodologic and qualitative information was extracted.

Data Synthesis  The information was synthesized into 2 sections: one describing the principles of different molecular diagnostic techniques, and the other highlighting the contributions of molecular diagnostic techniques to the understanding and diagnosis of several dermatologic diseases.

Conclusions  A basic understanding of the principles of molecular diagnostic techniques is crucial for the practicing dermatologist to benefit from the increasing number of molecular diagnostic articles appearing in the literature and potentially to apply these methods in clinical practice.

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Figure 1.

In G-banding, trypsin is used to partially digest histones and allow the chromosomes to relax and be dyed with Giemsa stain, which produces a distinctive banding pattern on each chromosome. A karyotype is generated that reveals any numeric or structural chromosomal abnormalities.

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Figure 2.

In fluorescence in situ hybridization, short, single-stranded DNA probes complementary to the DNA sequence of interest are created, fluorescently labeled, and hybridized with the target chromosomal DNA. A fluorescence microscope is used to detect the fluorescence generated in the regions where hybridization has occurred, allowing identification of the gene location and/or copy number changes present in the target chromosomes.

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Figure 3.

An illustration of the various types of fluorescence in situ hybridization (FISH). Blue circles represent interphase nuclei; stars, probes. A, In standard FISH, 2 differently labeled probes are generated and hybridized with interphase nuclei: one (yellow) complementary to the DNA sequence proximal to the break point of the involved chromosomes, and the other (red) complementary to the region distal to the translocation break point on the other chromosome involved in the translocation. In the normal cell, each normal allele displays its own single-color signal. In the abnormal cell where a reciprocal translocation has occurred, the derivative chromosome produces a dual-color fusion signal. B, In dual-fusion FISH, 2 sets of differentially labeled DNA probes complementary to sequences proximal and distal to the translocation break points on both chromosomes involved in the translocation (1 yellow, 1 red) are generated and hybridized with interphase nuclei. In the normal cell, strong, single-color signals (large stars) are produced by each normal allele. In the abnormal cell where reciprocal translocation has occurred, fused signals in both derivative chromosomes are displayed, with the remaining normal alleles showing a strong single-color signal. C, In break-apart FISH, 2 differentially labeled DNA probes complementary to sequences proximal (red) and distal (yellow) to the break point within 1 critical gene are generated and hybridized with interphase nuclei. In the normal cell, both copies of the selected gene are marked with a fusion signal (yellow and red). The abnormal cell, where the gene has been interrupted by translocation, displays 1 fusion signal for the normal allele and 2 single-color signals that have “broken apart” from each other, which serves to label each of the derivative chromosomes.

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Figure 4.

In comparative genomic hybridization, the ratio of differentially labeled sample (red) and reference (green) DNA hybridized to normal human metaphase chromosomes (blue) is graphically analyzed to detect copy number changes.

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Figure 5.

In the DNA microarray technique, segments of DNA in the form of complementary DNA (cDNA) or oligonucleotides serve as probes for detection and are arranged in a specific order on a nylon or glass support, which forms the microarray. Tissue sample RNA is harvested, reverse transcribed, labeled appropriately, and hybridized to the microarray slide. The different fluorescence generated by the sample and reference DNA is then measured and analyzed for differential expression. mRNA indicates messenger RNA; RT-PCR, reverse-transcriptase polymerase chain reaction.

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Figure 6.

In spectral karyotyping, 24 differentially labeled chromosome-specific probes are prepared by fractionating flow-sorted human chromosomes and labeling them with different combinations of fluorophores. The differentially labeled probes are then hybridized with metaphase preparations. They are visualized through a fluorescence microscope and analyzed by computer. The finished product is a color-coded karyotype in which each homologous chromosome pair has its own computer-assigned color, which allows identification of chromosomal translocations and rearrangements.

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