PMC3335511_01
Apparently balanced translocation is accompanied by a cryptic genomic deletion (a) Conventional karyotyping revealed a reciprocal translocation involving the short arm of one chromosome 11 (11p14) and the short arm of one chromosome 12 (12p13.2), which appears to be balanced. Chromosomes 16 appear normal. (b) Array CGH study detected a significant copy number loss on chromosome 16 (q23.2-q24.1) (c) This deletion was confirmed by FISH studies using a BAC probe RP11-625B13 (red) mapped to 16q23.3 and a control probe RP11-6C20 (green) mapped to 16p13.3. (d) Genes located within the deleted region 16q23.2-q24.1 (nucleotide 79,945,820 to 85,430,304, NCBI 36/HG 18) as shown by the UCSC genome browser. These include FOX family cluster FOXF1, FOXC2, and FOXL1.
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The followup G-banded chromosome study revealed a reciprocal translocation involving the short arm of one chromosome 11 and the short arm of one chromosome 12 - 46,XY,t(11;12)(p14;p13.2) (Figure 1(a)), which appeared to be balanced. Array CGH study was therefore performed using the CytoChip ISCA 8 x 60 K v2.0 Oligonucleotide array platform (BlueGnome) and showed that there is significant copy number loss (5.48 Mb in size) on the long arm of chromosome 16 (q23.2-q24.1) from nucleotide 79,945,820 to 85,430,304 (NCBI 36/HG 18) (Figure 1(b)). FISH studies using a BAC probe RP11-625B13 mapped to 16q23.3 (Figure 1(c)) and was not related to the translocation breakpoints. FISH analysis was also performed on parental blood samples which showed a normal result. Of note, this large deletion on chromosome 16 is not detectable on karyotype (Figure 1(a)).  This de novo deletion encompasses multiple genes including FOXF1, and two other related genes FOXC2 and FOXL1 (Figure 1(d)).
Complex rearrangement is revealed by array CGH. (a) FISH studies using acro-p probe targeting the short arms of all acrocentric chromosomes (red) were used to investigate the additional satellite on the long arm of 21qs. Insert shows the G-banded 21 chromosome with additional satellite on 21q (as indicated by *). (b) FISH studies using probes targeting 21q22 locus (encompassing RUNX1 gene, aqua) and 21qter locus (yellow) were used to lineate the deleted chromosome 21. The del(21) lacks the yellow signal for the 21qter locus and has a diminished signal for the 21q22 locus. The 4pter and 4qter probes were used because they are in the same cocktail with the 21q22 and 21qter probes (Abbott Molecular Inc. ToTelVysion Vial no.4 probe set). (c) By using the RUNX1/RUNX1T1 (previously known as AML1/ETO) dual color dual fusion translocation probe set (Abbott Molecular Inc), the FISH studies confirmed the diminished signal for the RUNX1 locus. (d) Array CGH study detected a complex rearrangement on the long arm of chromosome 21 with 3 copy number gains and 2 copy number losses. (e) The second copy number variation (highlighted by the broken line in (d)) contains multiple annotated genes including RUNX1, as shown by the UCSC genome browser.
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Cytogenetic investigations in the patient showed a chromosome abnormality with additional satellites on the distal long arm of one chromosome 21 (46,XY,21qs), which was confirmed by FISH studies using an Acro-p probe (Abbott Molecular Inc.) (Figure 2(a)). Further FISH studies demonstrated a deletion of the 21q subtelomere region (Figure 2(b)) with a diminished signal for the RUNX1 locus (Figure 2(c)), suggesting that one of the deletion breakpoints is located within the RUNX1 locus. Surprisingly, array CGH analysis using the Cytosure Syndrome Plus V2 2 x 105 k Oligonucleotide array platform (OGT) not only confirmed the complete deletion of the RUNX1 gene but it also revealed a complex chromosomal rearrangement of chromosome 21 with interspersed duplications (x3) and deletions (x2) (Figure 2(d)).  The second CNV is a loss which encompasses 1.81 Mb from nucleotide 34,965,815 to 36,781,907 and includes the gene RUNX1 (Figure 2(e)).
Array CGH identified a genomic loss and gain responsible for the patient's phenotype. (a) Patient's photo at the age of nine. His physical features demonstrate a relative macrocephaly with a prominent forehead, sparse hair and eyebrows, prominent ears which are low set, and cup-shaped, deep set eyes with esotropia, astigmatism, and possible congenital anomaly of the left optic disc. (b) Array CGH study detected a significant copy number loss on chromosome 4 (q13.2-q21.1). (c) Array CGH study also identified a significant copy number gain on chromosome 6 (q24.3). (d) The deletion was confirmed by FISH studies using a BAC probe RP11-165D10 (red) mapped to 4q13.2 and a control probe RP11-483A2 (green) mapped to 4q25. (e) The gain was confirmed by FISH studies using a BAC probe RP11-1077K2 (red) mapped to 6q24.3 and a control probe RP11-346K8 (green) mapped to 6p21.33. Of note, D and E are the same metaphase with different probe sets to visualize both chromosomes 4 and 6. The extra red signal appears to locate on the derivative chromosome 4. (f) Genes located within the deleted region 4q13.2-q21.1 (nucleotide 67,133,352 to 77,615,947, NCBI 36/HG 18) as shown by the UCSC genome browser. (g) Genes located within the duplicated region 6q24.3 (nucleotide 146,155,718 to 148,055,364, NCBI 36/HG 18) as shown by the UCSC genome browser.
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His physical features demonstrate a relative macrocephaly with a prominent forehead, sparse hair and eyebrows, prominent ears which are low set, and cup-shaped, deep set eyes with esotropia, astigmatism, and possible congenital anomaly of the left optic disc (Figure 3(a)). The first one is a copy number loss of a 10.5 Mb region on the long arm of chromosome 4 (q13.2-q21.1) from nucleotide 67,133,352 to 77,615,947 (Figure 3(b)). The second one is copy number gain of a 1.9 Mb region on the long arm of chromosome 6 (q24.3) from nucleotide 146,155,718 to 148,055,364 (NCBI 36/HG 18) (Figure 3(c)). Interestingly, the third copy of 6q24.3 was located at 4q13 (Figures 3(d) and 3(e)). The patient's clinical presentation is most likely due to the deletion and duplication of many genes involved in the imbalanced regions (Figures 3(f) and 3(g)).
FISH studies revealed a four-break balanced complex rearrangement in the patient's father. (a) By using BAC probe RP11-165D10 (red) and control probe RP11-483A2 (green), FISH studies identified that the 4q13.2 segment has moved from derivative chromosome 4 to derivative chromosome 6. (b) By using BAC probe RP11-1077K2 (red) and control probe RP11-346K8 (green), FISH studies identified that the 6q24.3 segment has moved from derivative chromosome 6 to derivative chromosome 4. (c) A simplified diagram to show the increased risk of having an unbalanced offspring in this family. Green represents the 4q13.2-q21.1 segment and the red represents the 6q24.3 segments. In the father, the green and red segment, switch locations leading to unbalanced offspring with either 3 copies of the red segment and 1 copy of the green segment (as in the patient) or 3 copies of the green segment and 1 copy of the red segment.
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The segment 4q13.2-q21.1 and the segment 6q24.3 are involved in a reciprocal insertional translocation which appears to be balanced (Figures 4(a) and 4(b)). The father now carries a significant risk of having another abnormal liveborn with either der(4)t(4;6) as in the patient or der(6)t(4;6) (Figure 4(c)).