The couple came to our attention due to the positive first trimester screen. The woman is a healthy 25-year-old, G1P0. The first trimester nuchal translucency screen at 13 weeks was positive, and the couple decided to proceed with chorionic villus sampling. To rule out common chromosomal aneuploidies, fluorescence in situ hybridization (FISH) studies were performed using specific probes targeting chromosome 13, 18, 21, X, and Y (AneuVysion kit, Abbott Molecular Inc.), and the results were normal. 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. To determine whether this translocation was familial, chromosome studies were performed on both parents and the results were normal. Subsequent prenatal ultrasounds showed signs of a significant cardiac defect as well as mild bilateral dilatation of the renal pelvis. Followup fetal echocardiogram confirmed the complex congenital heart defect:left ventricular hypoplasia, ventricular septal defect, and mild tricuspid insufficiency. Given the presence of the de novo reciprocal translocation, it was suspected that a cryptic imbalance may be present. 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)). This deletion was confirmed by 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)). 
The Database of Genomic Variants (DGV, NCBI 36/HG 18) is a useful tool to exclude benign CNVs. DGV revealed that there are no reported benign CNVs encompassing this deleted region, suggesting that it is not likely to be a benign change. This de novo deletion encompasses multiple genes including FOXF1, and two other related genes FOXC2 and FOXL1 (Figure 1(d)). Deletion in FOXF1 is associated with a lethal condition named alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV, OMIM no. 265380). Alveolar capillary dysplasia is characterized by abnormal development of the capillary vasculature around the alveoli leading to persistent pulmonary hypertension in the early postnatal period. Affected infants are usually born without any prior suspicion of lung malformation. However, most babies deteriorate rapidly, and many die of respiratory failure within the newborn period even with significant respiratory support. Other features of ACD/MPV include abnormal development of the cardiovascular, gastrointestinal, genitourinary, and musculoskeletal systems. A recent array CGH study demonstrated that alveolar capillary dysplasia is caused by the genomic deletion of the FOX gene cluster. It also suggests that the cardiovascular malformation in this disorder is caused by the haploinsufficiency for the neighboring FOXC2 and FOXL1 genes, as a patient with a downstream deletion encompassing just FOXC2 and FOXL1 exhibited cardiac defects without any respiratory complications. Therefore, this deletion is considered pathogenic and would explain the fetal cardiac anomalies seen on prenatal ultrasounds and fetal echocardiogram. The prognosis for this pregnancy is extremely poor with the presence of the lethal condition ACD in addition to the fetal cardiac anomalies. The previousy identified reciprocal translocation t(11;12) and this large deletion are most likely de novo events which occurred together within a single germ cell. Therefore, the recurrence risk will be low for future pregnancies. 
The majority of apparently balanced structural rearrangements are not associated with an abnormal phenotype. After the identification of such rearrangements, it is important to test the parents and determine whether it is inherited or de novo. Inherited rearrangements have reduced clinical concerns and are likely to be benign. However, de novo apparently balanced rearrangements predispose to a higher risk of a cryptic genomic imbalance at the breakpoints or somewhere else, which may be causative for the clinical phenotypes in the patients. Therefore, prenatal array CGH should be recommended in such circumstances for better clinical management. 
A 2-year boy was referred to the genetics clinic for assessment of his recurrent thrombocytopenia, short stature, and dysmorphic features. He was found to have isolated thrombocytopenia during acute illnesses which would resolve spontaneously with resolution of the intercurrent illness. Platelet count was below normal when well. Hematologic investigations included a normal peripheral blood smear, bone marrow aspirate and biopsy, and a normal immune workup. His past medical history was significant for vesicoureteral reflux, hypospadias, bilateral cryptorchidism, laryngomalacia, eczema, and constipation. He has mild fine motor and speech delays as well as behavioural issues including poor socialization, aggressive behaviour, and decreased attention. On physical examination at 6 years of age his growth parameters were all below the 3rd percentile. He had coarse hair, hypertelorism, epicanthal folds, short palpebral fissures, a depressed nasal root with a bulbous nasal tip, small teeth, low-set ears which were posteriorly angulated as well as short 5th fingers bilaterally with clinodactyly of the 5th digit on the left hand. Review of the family history revealed a younger brother who was small for age but was proportionate. He was nondysmorphic and had no other clinical concerns. Considering that he has a normal karyotype and his parents are small in stature, no further cytogenetic investigations (e.g., array CGH) were performed.
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)). To determine whether this abnormality was familial, chromosome studies were performed on both parents, and the results were normal. 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. Due to the clinical significance of haploinsufficiency for the RUNX1 gene (as discussed below), further investigation was undertaken to determine if the gene was completely deleted. 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 first and most proximal copy number variation (CNV) is a gain which encompasses a region of 1.1 Mb from nucleotide 23,449,744 to 24,557,710. DGV revealed there are no reported benign CNVs fully encompassing this duplicated region. However, there are no known genes within this region (by UCSC genome browser), suggesting that this duplicated region is of no clinical significance. 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)). No reported benign CNVs have been found encompassing this loss (by DGV). Besides RUNX1 which will be discussed later, there is another disease-associated OMIM gene CLDN14, which is associated with a type of autosomal recessive deafness. Since there is no concerns about the patient's hearing, haploinsufficiency of CLDN14 has unknown clinical significance. The third CNV is a gain encompassing 0.42 Mb from nucleotide 37,847,805 to 38,276,148. There is no reported benign CNVs fully encompassing this gain (by DGV), and there is only one RefSeq gene KCNJ6 located within it (by UCSC genome browser). Duplication of KCNJ6 is of unknown clinical significance. The fourth CNV is also a gain of 4.1 Mb in size from nucleotide 40,917,977 to 45,022,823. Similar to the other two gains, there is no reported benign CNVs fully encompassing this region (by DGV). UCSC genome browser revealed several disease-associated OMIM genes (TMPRSS3, CBS, CRYAA, CSTB, AIRE, and PFKL). Mutations and/or deletions of these genes are associated with various diseases. However, extra copies of them are of unknown clinical significance. The fifth and most distal CNV is a terminal loss encompassing 1.82 Mb from nucleotide 45,096,184 to 46,920,235. DGV showed no reported benign CNVs fully encompassing this loss, and UCSC genome browser revealed several disease-associated OMIM genes (as follows). Biallelic loss-of-function mutations in ITGB2, COL18A1, FTCD, and PCNT2 cause leukocyte adhesion deficiency (OMIM no. 116920), Knobloch syndrome type I (OMIM no. 267750), formiminotransferase deficiency (OMIM no. 229100), and type II icrocephalic osteodysplastic primordial dwarfism (OMIM no. 210720) respectively. Whether haploinsufficiency of these genes is associated with any clinical significance remains unknown. Mutations in COL6A1 and COL6A2 cause two autosomal dominant muscular diseases: Bethlem myopathy (OMIM no. 158810) and Ullrich congenital muscular dystrophy (OMIM no. 254090). Haploinsufficiency of the two genes could explain the 5th finger clinodactyly and the delay motor development observed in the patient. Of note, none of the gains encompass the Down syndrome critical regions. Besides, there is no probe between the fourth and the fifth CNVs and after the distal end of the fifth CNV.
 RUNX1, deleted in the second CNV, is a hematopoietic transcription factor frequently involved in somatic or acquired chromosome translocations associated with leukemia. The constitutional mutations and deletions of RUNX1 are known to be associated with familial platelet disorder with propensity to acute myelogenous leukemia (FPD/AML, OMIM no. 601399). FPD/AML is an autosomal dominant disorder which is characterized by the prolonged bleeding time, mild-to-moderate thrombocytopenia with normal platelet size and morphology, with or without abnormal platelet aggregation in response to arachidonic acid. Predisposition to the development of myeloid malignancies is another feature, with 20-50% progressing into AML and myelodysplasia. A lower cancer risk has been reported in individuals found to be haploinsufficient for RUNX1 while partial deletions or missense mutations have been associated with a higher risk for hematologic malignancy. By using the array CGH analysis, we confirmed that the patient was haploinsufficient for RUNX1 and thus has a decreased risk for AML and myelodysplasia. Furthermore, the complex rearrangement of chromosome 21 most likely represents de novo events. Therefore, the recurrence risk will be low for future pregnancies in this family.
This patient is the firstborn child to a healthy unrelated couple. At the time of birth the father and the mother's ages were 30 and 29, respectively. No anomalies were seen on the prenatal ultrasound, and the newborn was healthy. By nine months, the weight and length were at the 3rd percentile, while the head circumference remained at the 50th percentile. Developmental delay (delay in sitting, standing, and walking) was evident in the first year. At 16 months, severe expressive language delay was noted but receptive language was within normal limits. At 14 months, he had five seizures associated with fever and some focal signs. The patient was first seen at the genetics clinic at 20 months. No precise diagnosis had been made despite many investigations. The karyotype showed a normal male chromosome complement. Subtelomere FISH studies (TelVysion kit, Abbott Molecular Inc.) also showed a normal result. Other studies included UPD7, multiple biochemical tests, CT, brain MRI, skeletal survey, and thyroid studies which were all normal. At seven years nine months, the patient's height and weight were less then the 3rd percentile and head circumference remained at the 50th percentile. Now at the age of nine, he speaks intelligibly and in sentences. There are no behavior problems, no further seizures, and no sleep problems. He remains small despite a good appetite. 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)).
Once the technology was developed locally, array CGH was performed on the peripheral blood of the patient using the CytoChip ISCA 8 x 60 K v2.0 Oligonucleotide array platform (BlueGnome). The array analysis identified two chromosomal abnormalities. 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)). According to DGV, there are no reported benign CNVs fully encompassing this large deletion. The clinical significance of the deletion will be discussed later. 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)). DGV showed no reported benign CNVs fully encompassing this duplication, and UCSC genome browser revealed that there are no disease-associated OMIM genes within this region. In order to validate the array result, FISH studies were performed using a BAC probe RP11-135D10 mapping to the deleted region 4q13.2, a BAC probe RP11-1077K2 mapping to the duplicated region 6q24.3, two control probes on 4q25 (RP11-483A2) and on 6p21.33 (RP11-346K8) (RPC1-11 Human BAC library-CIHR Genome Resource Facility). Metaphase FISH confirmed the deletion at 4q13.2 and the duplication at 6q24.3. Interestingly, the third copy of 6q24.3 was located at 4q13 (Figures 3(d) and 3(e)). To further investigate whether this complex rearrangement is de novo or inherited, FISH studies were performed on the parental samples using the above 4 probes. The mother's result revealed a normal hybridization pattern with all probes hybridizing at the expected locations and with the expected copy number. The father's result, however, identified an abnormal hybridization pattern involving one chromosome 4 and one chromosome 6. 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)). Therefore, the patient's father has a four-break balanced complex rearrangement t(4;6)(q13.2q21.1;q24.3q24.3) which is not evident on the routine G-banded chromosomes. During the paternal meiosis, the der(4)t(4;6) and normal chromosome 6 segregated together and resulted in the chromosomal imbalances in this patient.
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)). However, there is a limited genotype-phenotype correlation in terms of each gene involved. The 10.5 Mb deletion on chromosome 4 contains 8 disease-associated OMIM genes (GNRHR, MUC7, ENAM, SLC4A4, GC, ALB, AFP, and SCARB2). Among these, ENAM mutations are associated with amelogenesis imperfecta, type 1B (OMIM no. 104500) which is an inherited defect of dental enamel formation with autosomal dominance as the likely mode of inheritance. However, this patient has a full set of primary teeth with normal tooth shape and enamel but delayed secondary teeth erupting. Currently, it is unclear whether haploinsufficiency of ENAM is responsible for the delay in secondary teeth erupting. The 1.9 Mb duplication on chromosome 6 does not contain any disease-associated OMIM genes (UCSC genome browser), and its clinical significance remains unknown.
Through combining array CGH and FISH techniques, we successfully identified the genomic imbalances responsible for the patient's clinical manifestation and further a familial chromosomal rearrangement which changes the predicted outcome and clinical management in this family. 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)). Therefore, prenatal diagnosis will be offered for any future pregnancies. The patient has two siblings who are both normal. Cytogenetic testing will be available to determine the carrier status when they become adults.