Diagnostic genome profiling in mental retardation

Abstract

Mental retardation (MR) occurs in 2%-3% of the general population. Conventional karyotyping has a resolution of 5-10 million bases and detects chromosomal alterations in approximately 5% of individuals with unexplained MR. The frequency of smaller submicroscopic chromosomal alterations in these patients is unknown. Novel molecular karyotyping methods, such as array-based comparative genomic hybridization (array CGH), can detect submicroscopic chromosome alterations at a resolution of 100 kb. In this study, 100 patients with unexplained MR were analyzed using array CGH for DNA copy-number changes by use of a novel tiling-resolution genomewide microarray containing 32,447 bacterial artificial clones. Alterations were validated by fluorescence in situ hybridization and/or multiplex ligation-dependent probe amplification, and parents were tested to determine de novo occurrence. Reproducible DNA copy-number changes were present in 97% of patients. The majority of these alterations were inherited from phenotypically normal parents, which reflects normal large-scale copy-number variation. In 10% of the patients, de novo alterations considered to be clinically relevant were found: seven deletions and three duplications. These alterations varied in size from 540 kb to 12 Mb and were scattered throughout the genome. Our results indicate that the diagnostic yield of this approach in the general population of patients with MR is at least twice as high as that of standard GTG-banded karyotyping.

Figure 1

Genomic profiles obtained by tiling-resolution array CGH for patients with MR. The arrays contained 32,447 human BAC clones (indicated by small circles representing the log₂-transformed and normalized test:reference intensity ratios [“2Log(T/R)”]), ordered from 1pter to Yqter in the two genome profiles and from pter to qter for individual chromosomes, on the basis of physical-mapping positions obtained from the UCSC Genome Browser (May 2004 freeze). A, Examples of de novo alterations, including deletions and duplications, in patients 1, 4, 5, 6, 8, and 9. B, Inherited copy-number variations in patients 117 and 498, including the matching chromosome profiles of one of their parents. The individual chromosome plots show small horizontal lines to indicate the presence of a copy-number alteration (gain shown in green; loss shown in red) detected by HMM analysis. Single-clone changes were ignored.

Figure 2

Frequency distribution of aneuploid segments per patient

Figure 3

Size distribution of chromosomal aberrations, divided into de novo alterations (n=10) and polymorphic variants (n=258). Black horizontal bars indicate median size (de novo alteration median 2.76 Mb; genomic variant median 0.43 Mb).

Figure A1

Details on patient 1, showing the genomewide array CGH profile, with the 3.93-Mb deletion on 1p34 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 1 (deletion indicated [red arrow in panel II]), confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III), and confirmation by FISH (panel IV) using clone RP11-708H15 (green) and a centromere probe as the control (red).

Figure A2

Details on patient 2, showing the genomewide array CGH profile, with the 0.92-Mb deletion on 2q23 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 2 (deletion indicated [red arrow in panel II]), confirmation of the de novo deletion by MLPA of patient DNA (panel III), and confirmation by FISH (panel IV) using clone RP11-469G04 (green).

Figure A3

Details on patient 3, showing the genomewide array CGH profile, with the 12.37-Mb deletion on 3q27-3q29 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 3 (deletion indicated [red arrow in panel II]), confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III), and confirmation by FISH (panel IV) using clone RP11-469G04 (red).

Figure A4

Details on patient 4, showing the genomewide array CGH profile, with the 1.24-Mb duplication on 5q35 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 5 (duplication indicated [red arrow in panel II]), and confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III).

Figure A5

Details on patient 5, showing the genomewide array CGH profile, with the 2.85-Mb deletion on 9q31 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 9 (deletion indicated [red arrow in panel II]), confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III), and confirmation by FISH (panel IV) using clone RP11-177I08 (green) and a centromere probe as the control (red).

Figure A6

Details on patient 6, showing the genomewide array CGH profile, with the 0.54-Mb deletion on 9q33 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 9 (deletion indicated [red arrow in panel II]), confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III), and confirmation by FISH (panel IV) using clone RP11-668H03 (red).

Figure A7

Details on patient 7, showing the genomewide array CGH profile, with the 7.49-Mb deletion on 11q14 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 11 (deletion indicated [red arrow in panel II]), confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III), and confirmation by FISH (panel IV) using clone RP11-118L16 (green) and a centromere probe as the control (red).

Figure A8

Details on patient 8, showing the genomewide array CGH profile, with the 2.3-Mb duplication on 12q24 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 12 (duplication indicated [red arrow in panel II]), and confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III).

Figure A9

Details on patient 9, showing the genomewide array CGH profile, with the interspersed 8.68-Mb duplication on 17p11-17p13 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 17 (duplication indicated [red arrow in panel II]), and confirmation of the de novo deletion by MLPA of both patient and parental DNA (panel III).

Figure A10

Details on patient 10, showing the genomewide array CGH profile, with the 2.66-Mb deletion on 22q11 (red arrow in panel I), the detailed array CGH profile of the affected chromosome 22 (deletion indicated [red arrow in panel II]), and confirmation of the de novo deletion by FISH (panel IV) using the LSI Tuple (Vysis) probe (red) and a centromere probe as the control (green).