In genetics, virtual karyotype is the digital information reflecting a karyotype, resulting from the analysis of short sequences of DNA from specific loci all over the genome, which are isolated and enumerated.Digital karyotyping – Wang et al., 10.1073/pnas.202610899 – Proceedings of the National Academy of Sciences It detects genomic copy number variations at a higher resolution for level than conventional karyotyping or chromosome-based comparative genomic hybridization (CGH). The main methods used for creating virtual karyotypes are array-comparative genomic hybridization and SNP arrays.
Background A karyotype (Fig 1) is the characteristic chromosome complement of a eukaryote species.White M.J.D. 1973. The chromosomes. 6th ed, Chapman & Hall, London. p28Stebbins G.L. 1950. Variation and evolution in plants. Chapter XII: The Karyotype. Columbia University Press N.Y. A karyotype is typically presented as an image of the chromosomes from a single cell arranged from largest (chromosome 1) to smallest (chromosome 22), with the sex chromosomes (X and Y) shown last. Historically, karyotypes have been obtained by staining cells after they have been chemically arrested during cell division. Karyotypes have been used for several decades to identify chromosomal abnormalities in both germline and cancer cells. Conventional karyotypes can assess the entire genome for changes in chromosome structure and number, but the resolution is relatively coarse, with a detection limit of 5-10Mb.
Method Recently, platforms for generating high-resolution karyotypes in silico from disrupted DNA have emerged, such as array comparative genomic hybridization (arrayCGH) and SNP arrays. Conceptually, the arrays are composed of hundreds to millions of probes which are complementary to a region of interest in the genome. The disrupted DNA from the test sample is fragmented, labeled, and hybridized to the array. The hybridization signal intensities for each probe are used by specialized software to generate a log2ratio of test/normal for each probe on the array.
Knowing the address of each probe on the array and the address of each probe in the genome, the software lines up the probes in chromosomal order and reconstructs the genome in silico (Fig 2 and 3).
Virtual karyotypes have dramatically higher resolution than conventional cytogenetics. The actual resolution will depend on the density of probes on the array. Currently, the Affymetrix SNP6.0 is the highest density commercially available array for virtual karyotyping applications. It contains 1.8 million polymorphic and non-polymorphic markers for a practical resolution of 10-20kb—about the size of a gene. This is approximately 1000-fold greater resolution than karyotypes obtained from conventional cytogenetics.
Virtual karyotypes can be performed on germline samples for constitutional disorders, and clinical testing is available from dozens of CLIA certified laboratories (genetests.org). Virtual karyotyping can also be done on fresh or formalin-fixed paraffin-embedded tumors. CLIA-certified laboratories offering testing on tumors include Creighton Medical Laboratories (fresh and paraffin embedded tumor samples) and CombiMatrix Molecular Diagnostics (fresh tumor samples).
Different platforms for virtual karyotyping Array-based karyotyping can be done with several different platforms, both laboratory-developed and commercial. The arrays themselves can be genome-wide (probes distributed over the entire genome) or targeted (probes for genomic regions known to be involved in a specific disease) or a combination of both. Further, arrays used for karyotyping may use non-polymorphic probes, polymorphic probes (i.e., SNP-containing), or a combination of both. Non-polymorphic probes can provide only copy number information, while SNP arrays can provide both copy number and loss-of-heterozygosity (LOH) status in one assay. The probe types used for non-polymorphic arrays include cDNA, BAC clones (e.g., BlueGnome ), and oligonucleotides (e.g., Agilent, or Nimblegen). Commercially available oligonucleotide SNP arrays can be solid phase (Affymetrix) or bead-based (Illumina). Despite the diversity of platforms, ultimately they all use genomic DNA from disrupted cells to recreate a high resolution karyotype in silico. The end product does not yet have a consistent name, and has been called virtual karyotyping, digital karyotyping, molecular allelokaryotyping, and molecular karyotyping. Other terms used to describe the arrays used for karyotyping include SOMA (SNP oligonucleotide microarrays) and CMA (chromosome microarray). Some consider all platforms to be a type of array comparative genomic hybridization (arrayCGH), while others reserve that term for two-dye methods, and still others segregate SNP arrays because they generate more and different information than two-dye arrayCGH methods.
Applications
Detecting copy-number changes Copy number changes can be seen in both germline and tumor samples. Copy number changes can be detected by arrays with non-polymorphic probes, such as arrayCGH, and by SNP-based arrays. Human beings are diploid, so a normal copy number is always two for the non-sex chromosomes. Deletions: A deletion is the loss of genetic material. The deletion can be heterozygous (copy number of 1) or homozygous (copy number of 0, nullisomy). Microdeletion syndromes are examples of constitutional disorders due to small deletions in germline DNA. Deletions in tumor cells may represent the inactivation of a tumor suppressor gene, and may have diagnostic, prognostic, or therapeutic implications. Gains: A copy number gain represents the gain of genetic material. If the gain is of just one additional copy of a segment of DNA, it may be called a duplication (Fig 4). If there is one extra copy of an entire chromosome, it may be called a trisomy. Copy number gains in germline samples may be disease-associated or may be a benign copy number variant. When seen in tumor cells, they may have diagnostic, prognostic, or therapeutic implications.
Amplifications: Technically, an amplification is a type of copy number gain in which there is a copy number >10. In the context of cancer biology, amplifications are often seen in oncogenes. This could indicate a worse prognosis, help categorize the tumor, or indicate drug eligibility. An example of drug eligibility is Her2Neu amplification and Herceptin, and an image of Her2Neu amplification detected by SNP array virtual karyotyping is provided (Fig 5).
Loss of heterozygosity (LOH), autozygous segments, and uniparental disomy Autozygous segments and uniparental disomy (UPD) are diploid/'copy neutral' genetic findings and therefore are only detectable by SNP-based arrays. Both autozygous segments and UPD will show loss of heterozygosity (LOH) with a copy number of two by SNP array karyotyping. The term Runs of Homozgygosity (ROH), is a generic term that can be used for either autozygous segments or UPD. Autozygous segment: An autozygous segment is bi-parental and seen only in the germline. They are extended runs of homozygous markers in the genome, and they occur when an identical haplotype block is inherited from both parents. They are also called "identical by descent" (IBD) segments, and they can be used for homozygosity mapping. Uniparental Disomy: UPD occurs when both copies of a gene or genomic region are inherited from the same parent. This is uniparental, in contrast to autozygous segments which are bi-parental. When present in the germline, they can be harmless or associated with disease, such as Prader-Willi or Angelman syndromes. Also in contrast to autozygosity, UPD can develop in tumor cells, and this is referred to as acquired UPD or copy neutral LOH in the literature (Fig 6). Acquired UPD is quite common in both hematologic and solid tumors, and is reported to constitute 20 to 80% of the LOH seen in human tumors. Acquired UPD can serve as the 2nd hit in the Knudson Two Hit Hypothesis of Tumorigenesis, and thus can be the biological equivalent of a deletion. Because this type of lesion cannot be detected by arrayCGH, FISH, or conventional cytogenetics, SNP-based arrays are preferred for virtual karyotyping of tumors. Figure 7 is a SNP array virtual karyotype from a colorectal carcinoma demonstrating deletions, gains, amplifications, and acquired UPD (copy neutral LOH).
Examples of clinical cancer applications A virtual karyotype can be generated from nearly any tumor, but the clinical meaning of the genomic aberrations identified are different for each tumor type. Clinical utility varies and appropriateness is best determined by an oncologist or pathologist in consultation with the laboratory director of the lab performing the virtual karyotype. Below are examples of types of cancers where the clinical implications of specific genomic aberrations are well established. This list is representative, not exhaustive. The web site for the Cytogenetics Laboratory at Wisconsin State Laboratory of Hygiene has additional examples of clinically relevant genetic changes that are readily detectable by virtual karyotyping.
Neuroblastoma Based on a series of 493 neuroblastoma samples, it has been reported that overall genomic pattern, as tested by array-based karyotyping, is a predictor of outcome in neuroblastoma: Tumors presenting exclusively with whole chromosome copy number changes were associated with excellent survival. Tumors presenting with any kind of segmental chromosome copy number changes were associated with a high risk of relapse. Within tumors showing segmental alterations, additional independent predictors of decreased overall survival were MYCN amplification, 1p and 11q deletions, and 1q gain.
Earlier publications categorized neuroblastomas into three major subtypes based on cytogenetic profiles: Subtype 1: favorable neuroblastoma with near triploidy and a predominance of numerical gains and losses, mostly representing non-metastatic NB stages 1, 2 and 4S. Subtypes 2A and 2B: found in unfavorable widespread neuroblastoma, stages 3 and 4, with 11q loss and 17q gain without MYCN amplification (subtype 2A) or with MYCN amplification often together with 1p deletions and 17q gain (subtype 2B).
Wilms' tumor Tumor-specific loss-of-heterozygosity (LOH) for chromosomes 1p and 16q identifies a subset of Wilms' tumor patients who have a significantly increased risk of relapse and death. LOH for these chromosomal regions can now be used as an independent prognostic factor together with disease stage to target intensity of treatment to risk of treatment failure.
Renal-cell carcinoma Renal epithelial neoplasms have characteristic cytogenetic aberrations that can aid in classification. See also Atlas of Genetics and Cytogenetics in Oncology and Haematology. Clear cell carcinoma: loss of 3p Papillary carcinoma: trisomy 7 and 17 Chromophobe carcinoma: hypodiploid with loss of chromosomes 1, 2, 6, 10, 13, 17, 21
Array-based karyotyping can be used to identify characteristic chromosomal aberrations in renal tumors with challenging morphology. Array-based karyotyping performs well on paraffin embedded tumors and is amenable to routine clinical use.
In addition, recent literature indicates that certain chromosomal aberrations are associated with outcome in specific subtypes of renal epithelial tumors.Clear cell renal carcinoma: del 9p and del 14q are poor prognostic indicators.Papillary renal cell carcinoma: duplication of 1q marks fatal progression.
Chronic lymphocytic leukemia