Article in press - uncorrected proof J Lab Med 2009;33(5):255–266 ᮊ 2009 by Walter de Gruyter • Berlin • New York. DOI 10.1515/JLM.2009.045 2009/45 Molekulargenetische und zytogenetische Redaktion: H.-G. Klein Diagnostik Array comparative genomic hybridisation in clinical diagnostics: principles and applications Array-CGH in der klinischen Diagnostik: Prinzipien und Anwendungen Uwe Heinrich1, Imma Rost1,*, Anthony Brown2, wertvollen, genom-weiten Screeningmethode zur Auf- Tony Gordon2, Nick Haan2 and Jessica Massie2 deckung chromosomaler Vera¨ nderungen in Form von Kopienzahlvarianten (CNV) entwickelt. Die kommerziell 1 Centre for Human Genetics and Laboratory Medicine erha¨ ltlichen Plattformen beinhalten die Subtelomerregio- Dr. Klein and Dr. Rost, Martinsried, Germany nen sowie die bekannten Mikrodeletions- und Mikrodu- 2 BlueGnome Ltd., Cambridge, UK plikationssyndromregionen, das restliche Genom wird mit unterschiedlichen Auflo¨ sungen von 8 kb bis 1 Mb abge- Abstract deckt. Neben der Aufdeckung eindeutig pathogener oder harmloser CNVs kann die aCGH auch CNVs mit unklarer In the last few years, array comparative genomic hybri- klinischer Signifikanz aufdecken, welche die Interpreta- disation (aCGH) has become a valuable genome-wide tion einer aCGH-Analyse erschweren. Ihre Hauptindi- screening tool for the detection of chromosomal aber- kationsstellungen umfassen Kinder mit mentaler rations in the form of copy number variants (CNVs). Com- Retardierung, Entwicklungsverzo¨ gerung, angeborenen mercially available platforms cover the subtelomeric Fehlbildungen und neuropsychiatrischen Erkrankungen regions and all known microdeletion/microduplication wie Autismus. In dieser Patientengruppe wird die aCGH syndrome regions, as well as the rest of the genome, with zunehmend die klassische Chromosomenanalyse als resolution ranging from 8 kb to 1 Mb. Besides detecting Ersttest ersetzen und ihr Einsatz in der Pra¨ nataldiagnos- clearly pathogenic or benign CNVs, aCGH can uncover tik steht derzeit in der Diskussion. Ein weiteres viel CNVs with unknown clinical significance, thus compli- versprechendes Einsatzgebiet ist die Tumordiagnostik, cating the interpretation of aCGH results. The main wo die aCGH dem Behandler die Klassifizierung und indications include children with mental retardation, Prognosestellung verschiedener Tumorentita¨ ten erleich- developmental delay, congenital anomalies and neuro- tern wird. psychiatric disorders such as autism. In this patient group, aCGH will gradually replace conventional chro- Schlu¨ sselwo¨ rter: Array-basierte genomische kompara- mosome analysis as the frontline test, and its implemen- tive Hybridisierung (aCGH); Kopienzahlvarianten (CNV); tation in prenatal diagnostics is in discussion. Another Mentale Retardierung; Mikrodeletionen, Mikroduplikatio- promising field is cancer diagnosis, for which aCGH will nen; Pra¨ nataldiagnostik. enable clinicians to classify and prognose different tumours more accurately in the near future. Keywords: array comparative genomic hybridisation Introduction (aCGH); copy number variant (CMV); mental retardation; microdeletions; microduplications; prenatal diagnostics. The first cytogenetic era started in 1956, when Tjio and Levan w1x determined for the first time that the exact number of human chromosomes was 46 using Giemsa- Zusammenfassung stained metaphase preparations (Figure 1). In the follow- In den letzten Jahren hat sich die Array-basierte geno- ing years, several syndromes were identified as being mische komparative Hybridisierung (aCGH) zu einer caused by numerical chromosome aberrations, such as Down syndrome (trisomy 21), Edward syndrome *Correspondence: Imma Rost, MD, Centre for Human (trisomy 18), Patau syndrome (trisomy 13) and Ullrich- Genetics and Laboratory Medicine Dr. Klein and Dr. Rost, Turner syndrome (monosomy X). Despite the very low Lochhamer Str. 29, 82152 Martinsried, Germany resolution of approximately 20–30 Mb, some structural Tel.: q49-89-8955780 Fax: q49-89-89557878 anomalies in the form of terminal deletions, such as dele- E-Mail: [email protected] tion 5p (cri du chat syndrome) and deletion 4p (Wolf- Article in press - uncorrected proof 256 Heinrich et al.: Array CGH Figure 1 Resolution of conventional and molecular cytogenetic techniques. (A) Partial karyotype showing a trisomy 21 corresponding to a duplication of 322 genes and 46 Mb of DNA. (B) Partial karyotype of a chromosome 22 pair and the corresponding ideograms at a 500-band level with a resolution of 4–6 Mb; the right chromosome 22 has a terminal 22q13.3 deletion (Phelan-McDermid syndrome); the deleted region has a size of ca. 6–8 Mb containing more than 100 genes. (C) FISH analysis of a metaphase showing a 22q11.2 microdeletion (DiGeorge syndrome) with the region-specific TUPLE1 probe in red and the control ARSA probe in green (the arrow denotes the missing red signal). The deleted region usually comprises 3 Mb and the TUPLE1 probe is 120 kb in length. In general, the maximum resolution of FISH depends on the probe length, ranging between 40 and 250 kb. (D) Interphase FISH representing a duplication of clone RP11-58H17 (green signals) in region Xp11.23 in a male patient. The red signal represents the X-centromere control probe DXZ1. (E) Result of an array CGH analysis corresponding to a 1.5-Mb deletion in band 15q13.3, a newly discovered recurrent microdeletion syndrome. The resolution of array CGH is platform- dependent, varying between 10 kb and 1 Mb. Hirschhorn syndrome), could be detected. The intro- imbalance could be detected in 2–7% of patients with duction of chromosomal banding techniques in the early idiopathic mental retardation (MR) and inconspicuous 1970s allowed genome analysis at a resolution of chromosome analyses w4, 5x. 5–10 Mb and led to the discovery of further deletion syn- The third era started in 1997–1998, when two groups dromes such as WAGR syndrome and Jacobsen syn- independently published the principle of array-based drome. High-resolution banding improved the resolution comparative genomic hybridisation (aCGH) w6, 7x. aCGH to 3–5 Mb and led to identification of the first micro- is based on co-hybridisation of the sample and reference deletion syndromes – Prader-Willi, DiGeorge and Smith- genomic DNA labelled with different fluorophores to Magenis syndromes. mapped DNA sequences or oligonucleotides that are The second era started in the mid-1980s, when fluoro- immobilised on a glass slide. The fluorescence of hybri- chrome-labelled DNA probes of 40–250 kb in length dised DNA is detected and expressed as a ratio that is dramatically increased the resolution w2x. With an plotted against genomic position so that an aberrant increase in the number of probes commercially available, region can be accurately mapped (Figure 2). Genomic this fluorescence in situ hybridisation (FISH) technique regions where there is more or less test sample relative rapidly became a standard method in the routine cyto- to the reference sample can be easily identified as a shift genetics laboratory, expanding the spectrum of micro- in the fluorescent ratio. The major difference with aCGH deletion syndromes identified (Williams-Beuren syndrome, compared to previous methods is replacement of the microdeletion 1p36) and revealing the first microdupli- metaphase spread with spotted DNA sequences. cation syndrome (microduplication 22q11.2). The great Although this modification precludes detection of purely disadvantage of FISH is the requirement for a tentative balanced translocations, it has the major advantage of pre-diagnosis, as FISH is a targeted diagnostic and not being a genome-wide screen at vastly improved resolu- a screening tool. A first step towards screening was intro- tion, limited only by the length of the DNA fragment spot- duced with the availability of a complete set of human ted and the genomic distance between DNA fragments. sub-telomeric probes to check for submicro- In addition, because only a DNA sample is required from scopic terminal deletions and cryptic balanced trans- a patient, much of the skill required for traditional band- locations w3x. Depending on the inclusion criteria, an ing karyotyping is removed. Using this method, copy Article in press - uncorrected proof Heinrich et al.: Array CGH 257 BAC arrays were the first to be explored and use large- insert (;200 kb) BAC clones spotted on glass slides. The major advantage of BAC arrays is that they give very robust results that are easy to interpret; this is because of the large regions of the genome included in each fea- ture. The primary disadvantage is that the resolution is limited by the size of the insert, so that even on high- density tiling arrays the maximum resolution that can be achieved is ;100 kb. This is in contrast to oligo arrays, which use short, generally 60-bp oligos so that the res- olution is only limited by the number of spots that can be physically fitted onto an array. The possible limitation with oligo arrays is that they produce noisier data because of the shorter hybridisation probe, so that smoothing (3–10 oligos) is necessary, reducing the effec- tive resolution. Nevertheless the results are good and enable detection of breakpoints down to the gene level. Recently produced oligo arrays include 1 million (Agilent Figure 2 Principle of array CGH. Technologies) and 2 million features (Roche Nimblegen Fluorescently labelled test and reference DNAs are co-hybridis- ed with Cot-1 to an array of spotted genomic DNA sequences Inc.) with an average probe