Large Clinically Consequential Imbalances Detected at the Breakpoints of Apparently Balanced and Inherited Chromosome Rearrangements  Sarah T. South, Lyndsey Rector, Emily Aston, Leslie Rowe, Samuel P. Yang  The Journal of Molecular Diagnostics  Volume 12, Issue 5, Pages 725-729 (September 2010) DOI: 10.2353/jmoldx.2010.090234 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Identical banding patterns with discordant microarray results in Patient 1 and his mother. A: Partial karyogram from Patient 1 showing chromosome 9 pair with abnormal chromosome 9 on right. Bracket on normal 9 shows region interpreted as inverted on abnormal 9. B: Genomic microarray ratio plots for chromosome 9 from Patient 1 showing gain of 12.2 Mb within 9q22–31.1 and loss of 7.2 Mb within 9q32–33.1. C: Partial karyogram from mother of Patient 1 showing chromosome 9 pair with identical banding pattern as observed in Patient 1. D: Genomic microarray ratio plots for chromosome 9 from mother of Patient 1 showing only a 550 kb deletion within 9q33.1. The Journal of Molecular Diagnostics 2010 12, 725-729DOI: (10.2353/jmoldx.2010.090234) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 FISH analysis of maternal chromosomes supports double intrachromosomal insertion and FISH analysis of Patient 1 supports noncontiguous duplication of 9q22–31.1 region. A: FISH on maternal chromosomes with a BAC probe to 9q31 (RP11–91L19) in green and a BAC probe to 9q32–33.1 (RP11–88F16) in orange and B: FISH with a BAC probe to 9q32–33.1 (RP11–88F16) in orange, and the ASS locus at 9q34.1 in aqua shows distal placement of 9q31 to 9qter and proximal placement of 9q32–33.1 with increased spacing between these regions on the derivative 9 and with no movement of 9q34.1. This signal pattern supports double insertion rather than inversion between q32 and q34.3. C: Diagram of location of 9q31, 9q32–33.1, and 9q34.1 probes on normal and derivative maternal chromosome 9s. D: FISH on Patient 1 chromosomes with a BAC probe to 9q22 (RP11–563G12) in orange and a BAC probe to 9q31 (RP11–91L19) in green, and the ASS locus at 9q34.1 in aqua shows the duplicated q22 region distal to the normally placed q31 region and not contiguous with the duplicated q31 region located at the distal 9qter region. E: Diagram of location of 9q22, 9q31, and 9q34.1 probes on normal and recombinant Patient 1 chromosome 9s. The Journal of Molecular Diagnostics 2010 12, 725-729DOI: (10.2353/jmoldx.2010.090234) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Proposed formation of imbalanced 9q in Patient 1 from recombination event in maternal meiosis. To allow maximal pairing between homologous regions on chromosome 9, a loop forms between q21 and q34, with q31 not participating in pairing. A recombination event within the loop then leads to a recombinant chromosome 9 with the observed duplications and deletions seen in the microarray analysis and in a pattern matching the FISH analysis. The Journal of Molecular Diagnostics 2010 12, 725-729DOI: (10.2353/jmoldx.2010.090234) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Identical banding patterns with discordant microarray results in Patient 2 and her mother. A: Partial karyogram from Patient 2 showing chromosome 1 pair and chromosome 5 pair with abnormal chromosomes on right of each pair. Bracket marks the region on chromosome 5 interpreted as inserted at the point of the arrow on chromosome 1. B: Genomic microarray ratio plots showing moving average for chromosomes 1, 5, and 6 from Patient 2 showing no imbalances of chromosome 1, a 10.5-Mb deletion within 5q33–34, and a 9.2-Mb duplication within 6p24–22. C: Partial karyogram from mother of Patient 2 showing chromosome 1 pair and chromosome 5 pair with identical banding pattern as observed in Patient 2. D: Genomic microarray ratio plots showing moving average for chromosomes 1, 5, and 6 showing no imbalances for these chromosomes. The Journal of Molecular Diagnostics 2010 12, 725-729DOI: (10.2353/jmoldx.2010.090234) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 FISH analysis of maternal chromosomes shows complex insertions between chromosomes 1, 5, and 6. A: FISH on maternal chromosomes with a BAC probe to 5q33 (RP11–79I6) in orange shows insertion of one copy of this region into 1q. B: FISH on maternal chromosomes with a BAC probe to 5q34 (RP11–90N23) in green shows insertion of one copy of this region into 6p. C: FISH on maternal chromosomes with a BAC probe to 6p23 (RP11–90N23) in orange shows insertion of one copy of this region into 1q. D: FISH with whole chromosome paints of 5 (orange) and 6 (red) on maternal chromosomes confirms insertion of chromosome 5 material into both chromosomes 1 and 6 and insertion of chromosome 6 material into chromosome 1, adjacent to inserted chromosome 5 material. E: Diagram of location of insertions of regions 5q33, 5q34, and 6p23 in maternal chromosomes. The Journal of Molecular Diagnostics 2010 12, 725-729DOI: (10.2353/jmoldx.2010.090234) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions